Western Blot Signals Research Articles

Lycibarbarspermidine L from the fruit of Lycium barbarum L. recovers intestinal barrier damage via regulating miR-195-3p

Ethnopharmacological relevanceThe fruit of Lycium barbarum L. is widely employed with the traditional effect of tonic properties. According to the theory of traditional Chinese medicine, Gou Qi can be distributed in the meridian of stomach, as well as the liver and kidney, indicating its effect on the digestive system. Clinical studies found that Gou Qi enhanced gastrointestinal functions. Pharmacological research showed the extract of Lycium barbarum exhibiting a repaired effect on the intestine barrier. Lycibarbarspermidine L (LBS L), which belongs to polyamines, is separated from the fruit of Lycium barbarum. However, it is unknown whether LBS L can restore damaged intestinal barrier like other polyamines such as spermidine. Aim of the studyTo elucidate the recovery effect of LBS L on damaged intestinal epithelium and its miRNA-related mechanism. Materials and methodsIEC-6 cells were used in vitro to assess the therapeutic effect of LBS L on the injured intestine and the regulation of miR-195-3p. Spermidine (SPD) with intestinal mucosal repair effect was used as a positive control. Sprague Dawley (SD) rats were subjected to 48 h fasting to induce intestinal epithelial atrophy in vivo. To determine the therapeutic effect of LBS L on injured intestinal epithelium and explore the mechanism, the fasting model group rats were treated with LBS L (25 mg/kg) for 4 days. ResultsResults in vitro showed that LBS L (10 μM) promoted cell proliferation and migration, affecting the S phase of the cell cycle. Western blot signals showed that LBS L raised the expression level of occludin. The miR-195-3p levels were decreased following LBS L treatment, which could be inversed by transfecting miR-195-3p mimic, demonstrating that LBS L inhibited miR-195-3p to improve cell growth. Results in vivo showed that LBS L could reverse the atrophic villi and inflammatory cell infiltration in the submucosa and restore miR-195-3p, occludin, and Ki67 levels in the intestine of mice in the fasting group. ConclusionsLBS L restores injured intestinal epithelium by reducing the expression of miR-195-3p.

Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems

PurposeIn vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems.MethodsQuiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period.ResultsFollowing entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro.ConclusionThis work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology.

Disbalance of the Duodenal Epithelial Cell Turnover and Apoptosis Accompanies Insensitivity of Intestinal Redox Homeostasis to Inhibition of the Brain Glucose-Dependent Insulinotropic Polypeptide Receptors in a Rat Model of Sporadic Alzheimer’s Disease

Introduction: Gastrointestinal dyshomeostasis is investigated in the context of metabolic dysfunction, systemic, and neuroinflammation in Alzheimer’s disease. Dysfunctional gastrointestinal redox homeostasis and the brain-gut incretin axis have been reported in the rat model of insulin-resistant brain state-driven neurodegeneration induced by intracerebroventricular streptozotocin (STZ-icv). We aimed to assess whether (i) the structural epithelial changes accompany duodenal oxidative stress; (ii) the brain glucose-dependent insulinotropic polypeptide receptor (GIP-R) regulates redox homeostasis of the duodenum; and (iii) the STZ-icv brain-gut axis is resistant to pharmacological inhibition of the brain GIP-R. Methods: GIP-R inhibitor [Pro3]-GIP (85 μg/kg) was administered intracerebroventricularly to the control and the STZ-icv rats 1 month after model induction. Thiobarbituric acid reactive substances (TBARSs) were measured in the plasma and duodenum, and the sections were analyzed morphometrically. Caspase-3 expression and activation were assessed by Western blot and multiplex fluorescent signal amplification. Results: Intracerebroventricular [Pro3]-GIP decreased plasma TBARSs in the control and STZ-icv animals and increased duodenal TBARSs in the controls. In the controls, inhibition of brain GIP-R affected duodenal epithelial cells, but not villus structure, while all morphometric parameters were altered in the STZ-icv-treated animals. Morphometric changes in the STZ-icv animals were accompanied by reduced levels of caspase-3. Suppression of brain GIP-R inhibited duodenal caspase-3 activation. Conclusion: Brain GIP-R seems to be involved in the regulation of duodenal redox homeostasis and epithelial cell turnover. Resistance of the brain-gut GIP axis and morphological changes indicative of abnormal epithelial cell turnover accompany duodenal oxidative stress in the STZ-icv rats.

Cytochrome P450 3A4 (CYP3A4) protein quantification using capillary western blot technology and total protein normalization

The western blot (WB) is the predominate method for protein quantification, frequently used in pharmacological and toxicological studies. To control for technical variation, WB signals are normalized through immunodetection of an internal standard “house-keeping” gene or total protein quantification via staining of the same blot or a duplicate, sister blot. Increasing evidence suggests that house-keeping genes are subject to change after drug treatment or under disease states, causing protein quantification errors in WB. Recent advances in automated capillary-based WB technologies enable measurement of the protein of interest, internal standards, and total protein in a single capillary. Using this approach, we quantified cytochrome P450 3A4 (CYP3A4) across 179 liver samples and compared normalization by both β-actin and total protein to determine which better functions as an internal standard. CYP3A4 is responsible for metabolism of a wide array of xenobiotics and is known to exhibit large inter-person variation, making it a good candidate to evaluate protein quantification. We observed significant differences in β-actin protein levels between liver samples (~20-fold) and found better correlation between CYP3A4 protein and mRNA using total protein normalization than β-actin, indicating total protein normalization to be less error prone for estimation of CYP3A4. Furthermore, by using total protein normalization, we confirmed significant association between CYP3A4 protein expression and the functional CYP3A4 variant CYP3A4*22, which contains two linked SNPs rs35599367 and rs62471956. Our results indicate that the automatic capillary WB instrument combined with total protein normalization provides a high throughput and robust approach for protein quantification.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

BirA*-protein A fusion protein based BioEnhancer amplifies western blot immunosignal.

Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin-AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity-dependent labeling ability, which could be used as a signal amplifier. We built a BirA*-protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity.

Overcoming off-targets: assessing Western blot signals for Bcnt/Cfdp1, a tentative component of the chromatin remodeling complex

The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the SNF2-related CBP activator protein (Srcap) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the Western blot signal against the mouse Bcnt/Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.

Noninvasive monitoring of the development and treatment response of ischemic hindlimb by targeting matrix metalloproteinase-2 (MMP-2).

Critical limb ischemia (CLI) is a common cause of high vascular morbidity and mortality. Monitoring the development and treatment response of hindlimb ischemia (HI) in an animal model enables a better understanding of the pathological mechanisms underlying CLI, and evaluation of the efficacy of novel therapeutic approaches. Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue including extracellular matrix degradation and angiogenesis. Herein, a mouse HI model is established and subjected to noninvasive optical imaging with a novel and ultra-sensitive MMP activatable probe, termed MMP-P12, for analyzing the development and treatment response of HI. Our results show that angiogenesis development during HI was well correlated with MMP-2 activity alteration as examined by western blot, histological staining and MMP-P12 fluorescence signal recovery. Moreover, vascular endothelial growth factor (VEGF) mediated HI treatment was also monitored by MMP-P12. Up-regulated MMP-2 expression and an enhancement of angiogenesis were observed after VEGF treatment, which peaked at 7 days after the treatment. Overall, our results showed that MMP-2 plays an important role in the monitoring of angiogenesis during HI development and therapy. Application of MMP-P12 to visualize MMP-2 activity alteration can serve as a promising noninvasive optical imaging strategy to monitor angiogenesis and its response to therapy in CLI.

Antigen retrieval by citrate solution improves western blot signal

In the present work, we describe and evaluate an additional step to the standard western blot protocol to increase signal strength after revealing. Weak or absence of signal is a common issue in western blot protocol leading to unexpected results. In our Antigen Retrieval for Western Blot Method (ARWB method), after transfer, the membrane was incubated in a citrate buffer following normal antigen retrieval procedure used for immunohistochemistry. Later, standard protocol was performed in order to reveal and compare with unexposed membranes to this antigen retrieval step. Signal in bands obtained by the modified protocol resulted significantly higher (in all 13 antibodies analyzed) compared to standard protocol. Some bands were only visible after citrate incubation. This method is a simple and economical way to improve results in western blot analysis.•The ARWB method significantly increases band’s density in all antibodies analyzed.•Protein localization does not influence the efficacy of the ARWB method since membrane and citoplasmatic proteins bands increase their signal in a similar way after the protocol is performed.•This ARWB method is simple, safe, economical and undoubtedly helpful in immunoblotting for proteins with weak signal.

Retraction: Intra-arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke mode.

Vibhuti, Khan R., Sharma A., Jain S., Mohanty S. and Prasad K. (2017) Intra-arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke model. J. Neurochem. 143, 722-735. https://doi.org/10.1111/jnc.14241.

Retraction Statement: Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways.

'Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways' by Slusarczyk, J., Trojan, E., Glombik, K., Piotrowska, A., Budziszewska, B., Kubera, M., Popiolek-Barczyk, K., Lason, W., Mika, J. and Basta-Kaim, A. The above article from the Journal of Neurochemistry published on 14 February 2016 on Wiley Online Library ( www.onlinelibrary.com), and in Volume 136, pp. 958-970, is being retracted by agreement between the corresponding author Agnieszka Basta-Kaim, the Journal's Editor-in-Chief Jörg Schulz, and John Wiley & Sons Ltd. The Editorial Office was alerted by a science journalist that the same Western Blot lane had been used to represent two different proteins. The Western Blot signal of iNOS in Fig.4a was supposedly identical to the Western Blot signal of phospho-JNK in Fig.6b. The corresponding author stated that "on the final step of figure 6 preparation the first author made, by mistake, an incorrect attachment of representative p-JNK blots." A corrected Fig.6b is enclosed below. The second concern reaching the Editorial Office was that the same Western Blot signal appeared to have been used to represent two different experimental conditions: the iNOS control signal (-/- LPS/TIA Fig.4a) appears as a horizontal and vertical mirror image of the last signal in this line (+/10 LPS/TIA Fig.4a). The raw membrane which was used to produce Fig.4a is enclosed on the next page and highlights the steps that were undertaken during figure preparation. Although the initial concern was not proven, concerns remained regarding the question how an inadvertent flipping of the first Western blot lane could happen. A corrected Fig.4a prepared by the corresponding author from the raw image of iNOS western blot depicted above, without flipped first lane, is presented below: Although the corresponding author provided a large amount of evidence to explain disparities in the presentation of Western Blot images, due to the number of inconsistencies that were revealed during review of the provided evidence and the inability to confirm the nature of the steps that led to them, it was felt that the above mentioned Western Blot images presented in this publication were not reliable, even if the conclusions may still be valid. The first author would like to apologize to the readers, reviewers and editors of Journal of Neurochemistry for the errors. Reference Slusarczyk J., Trojan E., Glombik K., Piotrowska A., Budziszewska B., Kubera M., Popiolek-Barczyk K., Lason W., Mika J. and Basta-Kaim A. (2016) Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways. J. Neurochem. 136, 958-970. https://doi.org/10.1111/jnc.13452.

Effect of post-mortem delay on N-terminal huntingtin protein fragments in human control and Huntington disease brain lysates.

Huntington disease is associated with elongation of a CAG repeat in the HTT gene that results in a mutant huntingtin protein. Several studies have implicated N-terminal huntingtin protein fragments in Huntington disease pathogenesis. Ideally, these fragments are studied in human brain tissue. However, the use of human brain tissue comes with certain unavoidable variables such as post mortem delay, artefacts from freeze-thaw cycles and subject-to-subject variation. Knowledge on how these variables might affect N-terminal huntingtin protein fragments in post mortem human brain is important for a proper interpretation of study results. The effect of post mortem delay on protein in human brain is known to vary depending on the protein of interest. In the present study, we have assessed the effect of post mortem delay on N-terminal huntingtin protein fragments using western blot. We mimicked post mortem delay in one individual control case and one individual Huntington disease case with low initial post mortem delay. The influence of subject-to-subject variation on N-terminal huntingtin fragments was assessed in human cortex and human striatum using two cohorts of control and Huntington disease subjects. Our results show that effects of post mortem delay on N-terminal huntingtin protein fragments are minor in our individual subjects. Additionally, one freeze-thaw cycle decreases the huntingtin western blot signal intensity in the cortex control subject, but does not introduce additional N-terminal huntingtin fragments. Our results suggest that subject-to-subject variation contributes more to variability in N-terminal huntingtin fragments than post mortem delay.

BlotCycler Consistently Shows an Increase in Western Blot Signal Intensity

BlotCycler Consistently Shows an Increase in Western Blot Signal Intensity

Abstract B26: Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin

Abstract Background: PARP inhibitors (PARPi) have been under clinical investigation as anti-cancer therapies both as monotherapy and in combination. The two basic mechanisms underlying PARP inhibitor activity are catalytic inhibition and PARP-1 trapping. PARPi trap PARP-1 onto base excision repair intermediates, contributing to potentiation of DNA damage by alkylating agents. The role of PARP-1 trapping with regard to efficacy and tolerability in the clinic is still under investigation. All PARPis are capable of PARP-1 trapping; however, different clinical-stage PARPi trap with dramatically different efficiencies. High efficiency trapping agents trap at doses similar to those required for catalytic inhibition while low efficiency trapping agents trap only at doses much higher than those required for inhibition. Being able to image trapped PARP-1 complexes in the cellular context would be beneficial for identifying other proteins associated with these complexes and may have clinical application in the detection of trapped PARP in tumor biopsies. To date, detection of PARP-1 complexes has been limited to low through-put, techniques that disrupt cellular architecture and lack the sensitivity to detect PARP-1 trapping in the absence of supraphysiologic doses of alkylating agents. More sensitive methods for quantifying trapped PARP1 complexes with spatial resolution are needed to enable further understanding of PARP-1 trapping in cancer and normal cells. The objective of this study was to develop an assay capable of identifying cellular PARP-1 trapped to chromatin with enhanced sensitivity and higher assay throughput. Methods: Duolink is a 96-well plate adaptable system that uses a proprietary antibody/oligonucleotide conjugate that, when localized within 40 angstroms, will anneal, amplify and hybridize to generate a quantitative proximity ligation assay (PLA) signal. To develop a Duolink trapping assay, antibodies to PARP-1, histone H2A.X and phosphorylated (Ser139) H2A.X were identified and optimized for labeling concentrations, endogenous target protein recognition and suitability for high content imaging. Duolink PLA was performed per the manufacturer's protocol. Automated image acquisition by Cell Insight and data analysis were performed using Cellomics View Software. PARP1 trapping was also evaluated by cellular fractionation and Western blot. Results: Binding to chromatin places PARP-1 in close proximity to histone H2A.X, presumably within the distance required for successful Duolink oligonucleotide annealing and amplification reactions. Co-treating cells with alkylating agents and PARPi induces a significant increase in phosphorylation of H2A.X (Ser139) as well as trapped PARP1 complexes. Comparative analysis of Western blot and PLA signals in HeyA8 cells treated with different PARPi/alkylating agent combinations revealed positive rank-order correlation in trapping activity. Resolution of in vitro single agent PARP-1 trapping by Western blot has been elusive but PLA detection of single agent PARP1 trapping was observed. Conclusion: The PLA trapping assay provides an improved methodology for evaluation of endogenous PARP-1 complexes bound to chromatin and may have utility for clinical tissue specimens where detection of PARP-1 trapping may lead to insights regarding efficacy and tolerability of PARP inhibitors. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Todd Hopkins, Barrett Ainsworth, Vivek Abraham, David Maag, Eric Johnson, Julie Wilsbacher. Development of a proximity ligation assay for the detection of PARP-1 trapped to chromatin [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B26.

Abstract P344: Increased Expression and Function of Endothelin Receptors in Aorta of (mRen2)27 Hypertensive Rats

Endothelin-1 (ET-1) contributes to various cardiovascular diseases including hypertension. ET-1 is produced in the endothelium and acts via ET A receptors, located in smooth muscle cells, and via ET B receptors, located in both endothelium and smooth muscle cells. Activation of ET A produces vasoconstriction, whereas endothelial ET B activation mediates vasodilation, with previous studies showing that ET-1-mediated vasoconstriction is enhanced in hypertension. We hypothesized that increased ETA-mediated function is present in vasculature of the (mRen2)27 hypertensive rat. Western blotting of total protein extracts from thoracic aorta was used to determine expression of ET A and ET B from Sprague-Dawley (SD n=4) and (mRen2)27 hypertensive (n=5) rats. Specificity of western blot signals for ET A and ET B was assessed by using pre-absorption of primary antibody with the corresponding antigenic peptide and intensity of signals was measured by densitometry (NIH-J Image). Contractile responses to ET-1 (10 -11 -10 -7 M) in intact and denuded aorta were determined by wire myography (Multi Myograph, DMT-USA) in the presence of the ET A blocker BQ123 (10 -6 M) or the ET B blocker BQ788 (10 -6 M). Contraction to ET-1 was expressed as maximal response (ET MAX as %K MAX ) and sensitivity (pD 2 =-Log [EC 50 ]). In aortae from (mRen2)27 rats ET A and ET B receptor expression (48 and 31 kDa bands) was greater relative to aortae of SD rats (p&lt;0.05). ET-1 contraction showed increased sensitivity in (mRen2)27 vs SD aortae (pD 2 , 8.17±0.15 vs 7.76±0.12, p&lt;0.05) with similar ET MAX (157±16 vs 145±6 %K MAX , p&gt;0.05). In intact arteries, blockade of ET B increased ET-1 sensitivity (pD 2 8.4±0.3, p&lt;0.05) in SD without effect on intact arteries from (mRen2)27 rats. In denuded arteries, ETB blockade increased ET MAX only in aortae from (mRen2)27 (205±12 vs 152±5, p&lt;0.05). Thus, increased ET A expression mediates greater ET-1-dependent contraction in vasculature of (mRen2)27 rats. In (mRen2)27 rats, loss of endothelial ET B receptor function in intact arteries may contribute to enhanced constrictor responses to ET-1, whereas increased ET B receptors in smooth muscle cells provide a counterbalancing vasodilation to offset maximal contractile effects of ET-1 in this model of hypertension.

A Study of Aβ Oligomers in the Temporal Cortex and Cerebellum of Patients with Neuropathologically Confirmed Alzheimer's Disease Compared to Aged Controls

Background/Aims: Investigations of Aβ oligomers in neuropathologically confirmed Alzheimer's disease (AD) are still scarce. We report neurohistopathological and biochemical analyses using antibodies against tau and amyloid β (Aβ) pathology. Methods: Thirty elderly AD patients and 43 age-matched controls with or without deposition of amyloid plaques (AP) were analyzed by immunohistochemistry. In 21 cases with available fresh tissue, Western blots were also performed. Neuropathological analysis included quantitative assessment of neurofibrillary tangles (NFT), AP and Aβ oligomer densities in the mesial temporal cortex (TC). Results: NFT, fibrillar amyloid and Aβ oligomeric deposit densities were significantly higher in AD patients than in controls. There was no relationship between oligomeric Aβ densities and Braak NFT staging scores. Furthermore, Aβ oligomer expression was closely correlated with Aβ plaques in the TC. By Western blot, Aβ oligomers were observed in AD patients, in plaque-free controls, in 1 ‘tangle-only AD' case, as well as in the cerebellum. A band near 55 kDa was the only Western blot signal that was significantly increased in the TC of AD patients compared to controls as well as less expressed in the cerebellum. Conclusion: These results suggest that a putative dodecamer, near 55 kDa, may contribute to AD vulnerability of the TC.

Proteomics-based identification of immunodominant proteins of Brucellae using sera from infected hosts points towards enhanced pathogen survival during the infection

Brucella (B.) species lack classical virulence factors, but escape effectively the immune response of the host. The species Brucella abortus and Brucella melitensis infect predominantly cattle and small ruminants such as sheep or goats, respectively, but account also for most human cases. These two species share remarkably similar genomes but different proteomes have been demonstrated. This might be one of the reasons for their host specificity. A comprehensive identification of immunodominant proteins of these two species using antibodies present in the serum of naturally infected ruminants might provide insight on the mechanism of their infection in different hosts. In the present study, whole-cell protein extracts of B. abortus and B. melitensis were separated using SDS–PAGE and western blotting was performed using field sera from cows, buffaloes, sheep and goats. Protein bands that matched with western blot signals were excised, digested with trypsin and subjected to protein identification using MALDI-TOF MS. Identified proteins included heat shock proteins, enzymes, binding proteins and hypothetical proteins. Antibodies against the same set of antigen were found for all species investigated, except for superoxide dismutase of B. melitensis for which antibodies were demonstrated only in sheep serum. Brucellae appear to express these proteins mainly for their survival in the host system during infection.

Cleaning Up Western Blot Signals From Immunoprecipitated Samples Using Alternative Detection Methods

High background signal is a common problem experienced when detecting proteins isolated through immunoprecipitation (IP) by Western blotting (WB). The most frequent cause of high background is signal interference from the heavy and light chain fragments of the denatured immunoprecipitating (or capture) antibody ' by-products of IP labeled by species-specific secondary antibodies at the WB stage. Here we comment on alternative methods for the detection of immunoprecipitated proteins by WB that avoid labeling of the heavy and light chain to varying extents. Certain methods have been described elsewhere (1), however, their use remains less widespread than traditional detection methods despite offering the researcher considerable advantages.

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.