Far Western Blotting Research Articles

Identification and characterization of HePP1, a Heart protein phosphatase 1 binding protein

We have conducted the search for the new protein phosphatase 1 (PP1)‐binding proteins of heart cells by using a yeast two‐hybrid system and identified six candidate proteins. One of those was a novel protein, Heart protein phosphatase 1 binding protein (HePP1). HePP1 contains 186 amino acids and is encoded by FLJ23654 . We have demonstrated that HePP1 was associated with PP1 in GST pull‐down assay, co‐immunoprecipitation, and far‐western blot. Northern blot analysis revealed that HePP1 mRNA was mainly distributed in heart and testis, but not in other tissues. The recombinant HePP1 cannot inhibit the PP1 activity, but antagonizes the inhibition of PP1 by phospho‐inhibitor‐1.

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.

Arabidopsis thaliana PRP40s are RNA polymerase II C-terminal domain-associating proteins

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions as a scaffold for RNA processing machineries that recognize differentially phosphorylated conserved (YSPTSPS) n repeats. Evidence indicates that proteins that regulate the phosphorylation status of the CTD are determinants of growth, development, and stress responses of plants; however, little is known about the mechanisms that translate the CTD phosphoarray into physiological outputs. We report the bioinformatic identification of a family of three phospho-CTD-associated proteins (PCAPs) in Arabidopsis and the characterization of the AtPRP40 ( Arabidopsis thaliana PRE-mRNA-PROCESSING PROTEIN 40) family as PCAPs. AtPRP40s-CTD/CTD-PO 4 interactions were confirmed using the yeast two-hybrid assay and far-Western blotting. WW domains at the N-terminus of AtPRP40b mediate the AtPRP40b-CTD/CTD-PO 4 interaction. Although AtPRP40s interact with both phosphorylated and unphosphorylated CTD in vitro, there is a strong preference for the phosphorylated form in Arabidopsis cell extract. AtPRP40s are ubiquitously expressed and localize to the nucleus. These results establish that AtPRP40s are specific PCAPs, which is consistent with the predicted function of the AtPRP40 family in pre-mRNA splicing.

Interaction of estrogen receptor‐α transactivation domain with nuclear proteins of mouse brain: p68 RNA helicase shows age‐ and sex‐specific change

Estrogen receptor (ER)-alpha interacts with nuclear proteins to mediate its multiple functions in the brain. However, it is not known which proteins interact with the ERalpha-transactivation domain (TAD) in mouse brain and whether they change with age and sex. Therefore, we have used affinity-purified GST-tagged mouse ERalpha-TAD fusion protein for interaction with nuclear proteins from the mouse brain. The pull-down assay and far-Western blotting detected four nuclear proteins of 100, 80, 68, and 50 kD. We have recently identified the 80-kD protein as MTA1 and demonstrated its decrease in old age. Here we report alteration in the interaction and expression of the 68-kD protein of adult and old mice of both sexes. This protein was identified as p68 RNA helicase through NCBI database search, immunoprecipitation, and immunoblotting. Further analysis showed that the extent of its interaction was relatively lower in old mice of both sexes and in male mice of both ages compared with their counterparts. However, the expression of p68 was significantly lower in old males compared with adult males, although other groups did not show significant changes. Such age- and sex-specific interaction of p68 suggests its implication in ERalpha-mediated brain functions during aging.

Avian pathogenic Escherichia coli bind fibronectin and laminin

Avian colisepticemia frequently occurs after respiratory tract damage, the primary site for infection allows bacteria to encounter an exposed basement membrane, where laminin and fibronectin are important components. We investigated the ability of an isolate of avian pathogenic Escherichia coli to bind fibronectin and laminin. Using Far-western dot blot analysis, we demonstrated the ability of this microorganism to bind basement membrane proteins fibronectin and laminin. Results from an ELISA-based approach indicate that the binding to these membrane proteins was bacterial-dose dependent. Furthermore, two specific E. coli polypeptides, of 32 kDa and 130 kDa, reacted with laminin and fibronectin, respectively. Further evaluation of these potential bacterial adhesins may provide insights into the pathogenesis of colibacillosis.

Four VirB6 Paralogs and VirB9 Are Expressed and Interact inEhrlichia chaffeensis-Containing Vacuoles

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to several virB genes and virD4 of Agrobacterium tumefaciens are found in an intravacuolar pathogen Ehrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size, E. chaffeensis has four tandem virB6 paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger than A. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction in E. chaffeensis. All four virB6 paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed by E. chaffeensis in THP-1 cells, and amounts of these five proteins were similar in isolated E. chaffeensis-containing vacuoles and vacuole-free E. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent in E. chaffeensis-containing vacuoles than in vacuole-free E. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest that E. chaffeensis VirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.

The C-terminal region of envelope protein VP38 from white spot syndrome virus is indispensable for interaction with VP24

White spot syndrome virus (WSSV) is a large, rod-shaped, enveloped double-stranded DNA virus. In this study, VP38, a viral envelope protein, was expressed as a glutathione S-transferase (GST) fusion protein, and a polyclonal antibody against VP38 was obtained. Far-Western blotting and GST pull-down showed that VP38 interacted directly with VP24, a major WSSV envelope protein. In addition, to delineate the interaction region of VP38 with VP24, GST-VP38n (aa 1-142) and GST-VP38c (aa 143-309) were expressed. The GST pull-down assay revealed that VP38 binds via its C-terminal region to VP24. The result implies that VP38 may participate in the formation of the WSSV envelope.

Interaction of Estrogen Receptor α Transactivation Domain with MTA1 Decreases in Old Mouse Brain

We have reported earlier that estrogen receptor (ER) alpha-transactivation domain (TAD) interacted with four nuclear proteins of 100 kD, 80 kD, 68 kD, and 50 kD of mouse brain and identified 68 kD as p68 RNA helicase and 50 kD as beta-tubulin. In this paper, we describe the identification of 80 kD nuclear protein as metastasis associated protein 1 (MTA1) and its interaction and expression in the brain of aging mice. Far-Western blotting and immunoprecipitation data revealed lower interaction of MTA1 in old than adult mice of both sexes. Furthermore, adult male showed lower expression of protein as compared to adult female. Altogether these findings suggest that age-dependent decrease in the expression of MTA1 and its interaction with ERalpha-TAD may influence the estrogen-mediated signaling pathway during aging of mouse brain.

Binding of theStreptococcus gordoniiDL1 Surface Protein Hsa to the Host Cell Membrane Glycoproteins CD11b, CD43, and CD50

Infective endocarditis is frequently attributed to oral streptococci. The mechanisms of pathogenesis, however, are not well understood, although interaction between streptococci and phagocytes are thought to be very important. A highly expressed surface component of Streptococcus gordonii, Hsa, which has sialic acid-binding activity, contributes to infective endocarditis in vivo. In the present study, we found that S. gordonii DL1 binds to HL-60 cells differentiated into monocytes, granulocytes, and macrophages. Using a glutathione S-transferase (GST) fusion to the NR2 domain, which is the sialic acid-binding region of Hsa, we confirmed that the Hsa NR2 domain also binds to differentiated HL-60 cells. To identify which sialoglycoproteins on the surface of differentiated HL-60 cells are receptors for Hsa, intrinsic membrane proteins were assessed by bacterial overlay and far-Western blotting. S. gordonii DL1 adhered to 100- to 150-kDa proteins, a reaction that was abolished by neuraminidase treatment. These sialoglycoproteins were identified as CD11b, CD43, and CD50 by GST pull-down assay and immunoprecipitation with each specific monoclonal antibody. These data suggest that S. gordonii DL1 Hsa specifically binds to three glycoproteins as receptors and that this interaction may be the initial bacterial binding step in infective endocarditis by oral streptococci.

TREM-2 binds to lipooligosaccharides of Neisseria gonorrhoeae and is expressed on reproductive tract epithelial cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is an innate immune receptor that initiates cellular activation upon ligation. In this study, we examined the interaction of TREM-2 with Neisseria gonorrhoeae using murine TREM-2A, as it has been reported to recognize bacterial ligands. Using a whole-bacteria enzyme-linked immunosorbent assay (ELISA), TREM-2A bound to all six strains in variable degrees. Far-western blots of gonococcal outer membranes revealed TREM-2A binding to lipooligosaccharide (LOS) and opacity (Opa) protein, with predominant binding to LOS. Binding of TREM-2A to LOS was confirmed by ELISA and surface plasmon resonance. O-deacylation of the lipid A significantly reduced binding. Flow cytometry and reporter cell assays showed that gonococci bound to TREM-2A-transfected cells and induced transmembrane signaling. In humans, TREM-2 was constitutively expressed by genitourinary and fallopian tube epithelial cells, both of which are primary targets of gonococcal invasion. Ligation of TREM-2 by LOS induced interleukin-6 production in HeLa cervical carcinoma cells. To our knowledge, this is the first report of the expression of human TREM-2 by cells deriving from a non-myeloid lineage. We conclude that gonococci can interact with TREM-2 receptors through binding to LOS and Opa protein and initiate cell signaling and cytokine production.

The nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. Using a yeast two-hybrid screen with the nucleocapsid (N) protein of SARS-CoV as a bait, the C terminus (amino acids 251 to 422) of the N protein was found to interact with human elongation factor 1-alpha (EF1alpha), an essential component of the translational machinery with an important role in cytokinesis, promoting the bundling of filamentous actin (F-actin). In vitro and in vivo interaction was then confirmed by immuno-coprecipitation, far-Western blotting, and surface plasmon resonance. It was demonstrated that the N protein of SARS-CoV induces aggregation of EF1alpha, inhibiting protein translation and cytokinesis by blocking F-actin bundling. Proliferation of human peripheral blood lymphocytes and other human cell lines was significantly inhibited by the infection of recombinant retrovirus expressing SARS-CoV N protein.

Direct protein–protein interaction of 11β-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11β-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17β-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11β-HSD1 but not with the related enzymes 11β-HSD2 and 17β-HSD2, suggesting a specific interaction between H6PDH and 11β-HSD1. The use of the 11β-HSD1/11β-HSD2 chimera indicates that the N-terminal 39 residues of 11β-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11β-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11β-HSD1. The protein–protein interaction and the involvement of the N-terminus of 11β-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11β-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11β-HSD1 and H6PDH involves a direct physical interaction of the two proteins.

Oligomerization of the potato virus X 25-kD movement protein

A 25-kD movement protein (25K protein) encoded by the first gene of the potexvirus Potato virus X triple gene block of transport genes is essential for the viral movement in infected plants. The 25K protein belongs to superfamily 1 of NTPase/helicases and exhibits in vitro RNA helicase, Mg2+-dependent NTPase, and RNA-binding activities. In the present work, the ability of 25K protein for homologous interactions was studied using the yeast two-hybrid system, protein chemical cross-linking in the presence of glutaraldehyde, far-Western blotting, and ultracentrifugation in sucrose density gradients. The 25K protein was shown to form homodimers and homooligomers. Sites of homologous protein-protein interactions were found in both the N- and C-terminal portions of the protein.

226. Characterisation of a putative mouse sperm - zona pellucida receptor complex

Mammalian spermatozoa acquire the ability to fertilise an oocyte as they ascend the female reproductive tract. This process is characterised by a complex cascade of biophysical and biochemical changes collectively known as capacitation. The attainment of a capacitated state is accompanied by a dramatic reorganisation of the surface architecture which renders the spermatozoa competent to recognise the heterogenetic matrix of the zona pellucida surrounding the oocyte and initiate fertilisation. Emerging evidence from our laboratory indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, to date the presence and composition of such a complex has yet to be described. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we have provided the first evidence that capacitated mouse spermatozoa express high molecular weight, multimeric protein complexes on their surface. Interestingly, at least two of these complexes contain heat shock protein 1 (HSPD1), a molecular chaperone that has previously been implicated in sperm-zona pellucida interaction. Furthermore, we were able to demonstrate that one of these complexes also possessed an affinity for solubilised zona pellucida as determined by Far-western blotting. 2D BN-PAGE was employed to further delineate the individual constituents of this high molecular weight complex, with several other chaperonin proteins not previously reported in functional sperm indentified. Collectively, these results support the notion the sperm-zona pellucida interaction are mediated by a multimeric receptor complex. Our current work is focussed on the identification of the key zona adhesion molecules that comprise this complex.

Simian Virus 40 DNA Replication Is Dependent on an Interaction between Topoisomerase I and the C-Terminal End of T Antigen

Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.

Interactions of an EssentialBacillus subtilisGTPase, YsxC, with Ribosomes

YsxC is a small GTPase of Bacillus subtilis with essential but still unknown function, although recent works have suggested that it might be involved in ribosome biogenesis. Here, purified YsxC overexpressed in Escherichia coli was found to be partly associated with high-molecular-weight material, most likely rRNA, and thus eluted from gel filtration as a large complex. In addition, purification of ribosomes from an E. coli strain overexpressing YsxC allowed the copurification of the YsxC protein. Purified YsxC was shown to bind preferentially to the 50S subunit of B. subtilis ribosomes; this interaction was modulated by nucleotides and was stronger in the presence of a nonhydrolyzable GTP analogue than with GTP. Far-Western blotting analysis performed with His(6)-YsxC and ribosomal proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that YsxC interacted with at least four ribosomal proteins from the 50S subunit. Two of these putative protein partners were identified by mass spectrometry as L1 and L3, while the third reactive band in the one-dimensional gel contained L6 and L10. The fourth band that reacted with YsxC contained a mixture of three proteins, L7/L12, L23, and L27, suggesting that at least one of them binds to YsxC. Coimmobilization assays confirmed that L1, L6, and L7/L12 interact with YsxC. Together, these results suggest that YsxC plays a role in ribosome assembly.

Monoclonal antibody against Rab8 from Bombyx mori (Lepidoptera: Bombycidae)

Small GTPases of the Rab family are key regulators of membrane trafficking. Monoclonal antibodies are useful tools for identifying proteins that interact with other proteins and for examining their tissue distribution. We selected a monoclonal antibody against Rab8 of Bombyx mori L. It specifically recognized amino acid residues 30-109, which are conserved among Rab8 proteins, and did not recognize any other Rab proteins. Western blotting using the antibody revealed one band in the brains of B. mori and rat. Far-Western blotting analysis detected three proteins interacting with Rab8. These results indicate that this antibody is useful for clarifying the physiological function of Rab8 of B. mori and other species. This is a report of a study on a monoclonal antibody against insect Rab protein.

Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.

P40 phox as an alternative organizer to p47 phox in Nox2 activation: A new mechanism involving an interaction with p22 phox

p40 phox activated phagocyte NADPH oxidase without p47 phox in a cell-free system consisting of p67 phox, Rac and cytochrome b 558 relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47 phox. Addition of p47 phox slightly increased the activation, but not additively. p40 phox improved the efficiency of p67 phox in the activation. The C-terminus-truncated p67 phox, p40 phox(D289A), p40 phox(R58A), or p40 phox(W207R) showed an impaired activation. A peptide corresponding to the p22 phox Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40 phox SH3 domain. Thus, p40 phox can substitute for p47 phox in the activation, interacting with p22 phox and p67 phox through their specific regions.

Far Western: Probing Membranes

INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.