90-kDa Protein Research Articles

Evaluation of haemolymph phenoloxidase activity from the grub of Zophobas morio as a predictor of immune response.

In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30°C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85kDa and 55kDa polypeptides in all the experiments over control after 24h, 48h and 96h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55kDa protein and 85kDa protein. A significant increase in 85kDa polypeptide was observed in the haemolymph of the grubs after 72h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.

Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125kDa) and X2 variants (88 and 79kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

In vitro Gluten Degradation Using Recombinant Eurygaster Integriceps Prolyl Endoprotease: Implications for Celiac Disease.

Celiac disease (CD) is a gluten-sensitive chronic autoimmune enteropathy. A strict life-long gluten-free diet is the only efficient and accepted treatment until now. However, maintaining a truly gluten-free status is both difficult and costly, often resulting in a social burden for the person. Moreover, 2 to 5 percent of patients fail to improve clinically and histologically upon elimination of dietary gluten. Therefore, novel therapeutic approaches, including gluten degrading enzymes, are an unmet need of celiac patients. To evaluate the function of sunn pest prolyl endoprotease for gluten and gliadin hydrolysis in vitro. The spPEP was expressed as a recombinant protein in E. coli BL21 (DE3), and its catalytic activity was assessed by SDS-PAGE and RP-HPLC analyses. Production of a 100-kDa spPEP protein was confirmed by SDS-PAGE and western blot analysis. Also, we demonstrate that spPEP efficiently degrades gluten and α-gliadin (30-40 kDa) in vitro under conditions similar to the GI and is resistant to pepsin and trypsin. The gathered data demonstrated that spPEP might be a novel candidate for Oral Enzymatic Therapy (OET) in CD and other gluten-related disorders.

34-kDa salivary protein enhances duck Tembusu virus infectivity in the salivary glands of Aedes albopictus by modulating the innate immune response

Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors.

Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Type I-like metalloproteinase in the venom of the West African saw-scaled carpet viper (Echis ocellatus) has anti-trypanosomal activity against African trypanosomes

African trypanosomiasis is an infectious disease caused by hemoparasites of the genus Trypanosoma and remains a major health problem in Africa - killing around 4000 people and animals worth an estimated $5 billion, annually. The absence of a vaccine and satisfactory drug against African trypanosomiasis (AT) necessitates the continued search for new chemotherapy options. Owing to the rich biochemical diversity in snake venom, it has recently become a source of therapeutic peptides that are being explored for the development of novel drug candidates for diverse ailments such as cancers and infectious diseases. To explore this, Echis ocellatus venom (EOV) was investigated for the presence of an anti-Trypanosoma factor, with the subsequent aim to isolate and identify it. Crude EOV was collected and tested in vitro on the bloodstream form (BSF) i.e. long and slender morphological form of Trypanosoma brucei and T. congolense. This initial testing was followed by a sequential anti-trypanosomal assay guided purification of EOV using ethanol precipitation, distillation, and ion exchange (IEX) chromatography to obtain the active trypanocidal component. The purified anti-Trypanosoma factor, estimated to be a 52-kDa protein on SDS-PAGE, was subjected to in-gel trypsin digestion and 2D RP HPLC-MS/MS to identify the protein. The anti-Trypanosoma factor was revealed to be a zinc-dependent metalloproteinase that contains the HEXXHXXGXXH adamalysin motif. This protein may provide a conceptual framework for the possible design of a safe and effective anti-trypanosomal peptide for the treatment of AT.

Identification of CRKL as an oncogenic biomarker for prognosis and immunotherapy in melanoma, and its potential molecular mechanism

CRKL (CRK Like Proto-Oncogene) belongs to the Crk family and is a 39-kDa adapter protein that encodes SH2 and SH3 (src homologs) domains. To identify its oncogenic role in malignant melanoma, we investigated the association between CRKL and mutation, prognosis, tumor mutation burden, immune cell infiltration of melanoma, and explored the associations between CRKL and immunotherapy response. Our results showed that abnormal CRKL expression is associated with poor prognosis in melanoma and is significantly correlated with immune-activated pathways and processes, immune cell infiltrations, and expression of immunoregulators. Importantly, we found that CRKL expression is a predictive biomarker for anti-PD1 therapy response in melanoma patients. Furthermore, inhibiting CRKL expression in melanoma cell lines suppressed their proliferation and metastasis, as well as activated the pyroptosis-related pathway. Our study provides potential mechanisms of melanoma pathogenesis, which may suggest new avenues for targeted therapy in this disease.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

A viral biomolecular condensate coordinates assembly of progeny particles.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes1,2. Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation3-8. Although linked to several viral processes3-5,9,10, evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection.

Expressing recombinant human lactoferrin with antibacterial activity in Nicotiana benthamiana.

Lactoferrin is a non-hematic iron-binding 80-kDa protein that exhibits antimicrobial activity. Higher plants function as "green bioreactors" for large-scale recombinant protein production. In this study, we transiently expressed recombinant human lactoferrin (rhLF) in Nicotiana benthamiana at a yield of approximately 40 µg g-1 fresh mass (gFM) using the Tsukuba system. Additionally, the expression level of rhLF increased when it was fused with KDEL, an endoplasmic reticulum retention motif. Purified plant-derived rhLF possesses antibacterial activity that inhibits the growth of Escherichia coli. These results indicated that rhLF containing antimicrobial activity can be produced in N. benthamiana using the Tsukuba system.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in rainbow trout (Oncorhynchus mykiss).

Renibacterium salmoninarum is a pathogenic gram-positive bacterium and the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in rainbow trout (Oncorhynchus mykiss). The CK-90 strain was previously isolated in steelhead trout (O. mykiss) from Dworshak National Fish Hatchery (NFH) in 1990. It was intracoelomically injected (100 μL) at a high dose containing 4.80 x 106 CFU g fish-1 (OD525 =1.779) and a low dose containing 6.86 x105 CFU g fish-1 (OD525 = 1.077), or fish were immersed in a solution containing 2.21 x 105 CFU g fish-1 (OD525 =0.886) of R. salmoninarum. The ATCC 33739 strain, originating from brook trout (Salvelinus fontinalis), was also included and intracoelomically-injected at 3.58 x 105 CFU/g (OD525 =1.431) to discern differences in virulence between the strains. Clinical signs of BKD manifested approximately ten days post-challenge, and mortalities began 19 days post-challenge. To confirm infection and quantify R. salmonirarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected post-challenge. Injection-challenged rainbow trout with CK-90 (both high and low groups) exhibited significantly higher mortality (P<0.001) than fish injected with ATCC 33739 or those exposed to CK-90 via immersion challenge. The R. salmoninarum antigen, p57-kDa, was confirmed via ELISA. Antigen load for fish injected with the CK-90 strain (high dose: OD405 =0.71, low dose: OD405 =0.66) was significantly higher than for fish injected with the ATCC 33739 strain (OD405 =0.34). It was found that the CK-90 strain (both high and low doses) was more virulent than ATCC 33739 strain, which exhibited no mortalities over 28 days. Although there were no mortalities during the 28-day trial, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in ATCC 33739 fish. Further, the immersion challenge methodology for these R. salmoninarum strains was ineffective in inducing mortalities at the abovementioned doses.

SDS-PAGE analysis of hormone treated larvae of honeybee Apis cerana indica (F.) (Hymenoptera: Apidae)

SDS-PAGE analysis of protein was carried out in fifth instar larvae of worker and drone caste at 24 hrs and 36 hrs treatment with acetone (control), JH-III and 20-HE. 52kDa to 106 kDa protein bands were observed in both caste of larvae after JH-III, 20-HE and acetone treatment after 24 hrs and 36 hrs. Female specific 170kDa vitelogenin like protein band was observed in larvae of both caste. Caste specific protein band 191kDa and 32kDa was observed in worker larvae and 38kDa protein band was observed in drone larvae. This difference is due to different developmental timing and difference in physiology of both caste.

The immunoexpression of valosin-containing protein and small VCP-interacting protein in rat ovaries.

Numerous cellular processes are controlled by the ubiquitin-proteasome-mediated degradation pathway, involve the 97-kDa valosin-containing protein (p97/VCP). Small p97/VCP-interacting protein (SVIP) was first discovered as one of the novel androgen-responsive genes as well as one of the many cofactors controlling p97/VCP. The aim of the study was to investigate localization and immunoexpression of p97/VCP and SVIP in rat ovarian tissue. The histomorphological examination of rat ovarian tissue was performed by using haematoxylin-eosin (HE) staining. Using the immunohistochemical technique, cellular location and expression of p97/VCP and SVIP in rat ovarian tissue were examined. The nuclear and cytoplasmic immunoexpression of p97/VCP and SVIP was observed in the different stages of ovarian follicles and corpus luteum in the rat ovaries. The immunolocalization of SVIP and VCP in the rat ovaries suggest that they may be involved in the oogenesis. Further studies should be performed about the function of the VCP and SVIP in the female reproductive tract.

Transcriptional regulation of spatiotemporal gene expression within the seminiferous epithelium: Mouse Acrv1 gene as a model.

Precise spatiotemporal expression of cohorts of differentiation markers unique to spermatogonia, spermatocytes, and round spermatids punctuates spermatogenesis and ensures its completion. For example, genes coding for the synaptonemal complex or the acrosome or flagellum areexpressed sequentially in a developmental stage- and germ cell-specific manner. But the transcriptional mechanisms governing the spatiotemporal order of gene expression within the seminiferous epithelium are poorly understood. Using the round spermatid-specific Acrv1 gene, which codes for the acrosomal protein SP-10 as a model, we learned that (1) the proximal promoter itself contains all the necessary cis-regulatory sequences, (2) an insulator prevents somatic cell expression of the testis-specific gene, (3) RNA II polymerase is loaded on the Acrv1 promoter but paused in spermatocytes, thus ensuring precise transcriptional elongation in round spermatids, and that (4) a transcriptional repressor binding protein of 43 kilodaltons (TDP-43) plays a role inmaintaining the paused state in spermatocytes. Although the Acrv1enhancer element has been narrowed down to 50bp and its binding to a 47kDa testis-abundant nuclear protein shown, the identity of the putative transcription factor responsible for activation of round spermatid-specific transcription remains elusive. Human male infertility is idiopathic with limited treatment options. Understanding transcriptional regulation of spermatogenesis has the potential to lead to future therapies for male infertility.

Feeding Colostrum Rapidly Alters Enzymatic Activity and the Relative Isoform Abundance of Jejunal Lactase in Neonatal Pigs ,

Our objective was to determine the short-term effects of feeding colostrum and mature milk on the enzyme activity and relative isoform abundance of lactase in neonatal pigs. We studied newborn pigs that were fed colostrum, mature milk and water for 6 h (Study 1). We also studied unfed pigs shortly after birth, and newborn pigs fed either colostrum or mature milk for 24 h (Study 2). The specific activity (micromol x min(-1) x g protein(-1)) of lactase in ajejunal mucosal homogenate and a purified membrane fraction was lower in pigs fed colostrum than in unfed newborns or those fed either milk or water. However, after 24 h, total jejunal lactase activity (micromol x mol(-1)), jejunal mass and protein content were higher in the colostrum- and milk-fed pigs than in the unfed newborns. In colostrum-fed pigs, the reduction in lactase specific activity after 6 h was associated with 1) a marked increase in the relative abundance of a 180-kDa protein, which was shown to be one of three pro-lactase isoforms, and 2) a lower relative abundance of the 160-kDa isoform, considered to be the mature form of the enzyme. Our evidence suggests that feeding either colostrum or mature milk increases total jejunal lactase activity. The reduction in both the specific activity and abundance of the mature isoform in conjunction with an increased relative abundance of the 180-kDa pro-lactase isoform suggests that feeding colostrum alters the post-translational processing of intestinal lactase in neonatal pigs.

Haemolymph Proteins on Stressed Cockroach Periplaneta Americana

Immune responses in insects are humoral and cell mediated and the haemolymph proteins are found to be involved in humoral immune responses. In this experiment cockroaches were divided into two groups as control and wounded and the haemolymph samples were collected at different time intervals and subjected for quantitative analysis of proteins, proteolytic activity and protein profile. Haemolymph protein concentration reduced initially after injury then they recovered after 24 hrs. Proteolytic activity was found to be the highest during the first 24hrs of the wounded insects. Seven protein bands were observed in PAGE gel electrophoresis of the wounded and control cockroaches. A 115 kDa protein band was found to appear in the wounded P.americana after 8 hours is an important finding of this experiment. Some of the protein bands were reduced in the wounded cockroach and another important finding was protein retaining to normal level in the haemolymph after 24hrs and 80 and 76kDa protein bands were thicker in this group. Cockroach recovering after wounding is revealed after 8hrs after wounding.

Ligand binding and pH influence conformational flexibility of domains I and III in bovine serum albumin.

Bovine Serum Albumin (BSA) is a multi-functional protein that changes its conformation in response to pH and has a high binding capacity for various ligands. Due to these properties, BSA can be used in immunoassay and cell culture applications. To understand how BSA may affect assay function, it is important to understand the flexibility and dynamics of BSA and how it can influence these applications. BSA is a 66-kDa protein composed of three structurally homologous domains. Ligand binding sites can be affected by change in pH as it nears the isoelectric point 4.7-5.2. In our study, we focused on how pH and caprylate impact conformational flexibility of BSA. Using molecular dynamics (MD), we performed simulations using Amber on BSA at pH 7.0 and pH 5.2 both ligand-free and caprylate-bound. We compared all four structures using the root mean square deviation and observed that both the ligand-free and caprylate-bound at pH 7.0 had more structural variation over time than at pH 5.2. The root mean square fluctuation was calculated to compare the conformational flexibility between the residues. We found there were significant conformational differences in Domain I and Domain III at pH 7.0 than pH 5.2 for both ligand-free and caprylate-bound BSA. The surface charge in Domain I is significantly affected with change in pH that may impact the interactions with Domain III and alter binding sites. BSA is a dynamic protein that was found to be structurally different using MD. By understanding BSA conformational flexibility, we can have better insight on its functionality in immunoassay applications.

LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma.

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion invitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, invivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

The NLRP6 protein is very faintly expressed in several normal and cancerous epithelial cells and may be confused with an unrelated protein.

Nod-Like Receptor Pyrin domain-containing protein 6 (NLRP6), a member of the Nucleotide-oligomerization domain-Like Receptor (NLR) family of proteins, assembles together with the ASC protein to form an inflammasome upon stimulation by bacterial lipoteichoic acid and double-stranded DNA. Besides its expression in myeloid cells, NLRP6 is also expressed in intestinal epithelial cells where it may contribute to the maintenance of gut homeostasis and negatively controls colorectal tumorigenesis. Here, we report that NLRP6 is very faintly expressed in several colon cancer cell lines, detected only in cytoplasmic small dots were it colocalizes with ASC. Consequently, it is very hardly detected by standard western-blotting techniques by several presently available commercial antibodies which, in contrast, highly cross-react with a protein of 90kDa that we demonstrate to be unrelated to NLRP6. We report here these results to caution the community not to confuse the 90kDa protein with the endogenous human NLRP6.