scholarly journals Validation of antibodies for the specific detection of human TRPA1

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
H. S. Virk ◽  
M. Z. Rekas ◽  
M. S. Biddle ◽  
A. K. A. Wright ◽  
J. Sousa ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Airway Smooth Muscle Cells ◽  
Published Data ◽  
Specific Detection ◽  
Flow Cytometry Assay ◽  
Transient Receptor ◽  
Antibody Validation ◽  
Negative Cell Line

AbstractThe transient receptor potential cation channel family member ankyrin 1 (TRPA1) is a potential target for several diseases, but detection of human TRPA1 (hTRPA1) protein in cells and tissues is problematic as rigorous antibody validation is lacking. We expressed hTRPA1 in a TRPA1-negative cell line to evaluate 5 commercially available antibodies by western blotting, immunofluorescence, immunocytochemistry and flow cytometry. The three most cited anti-TRPA1 antibodies lacked sensitivity and/or specificity, but two mouse monoclonal anti-TRPA1 antibodies detected hTRPA1 specifically in the above assays. This enabled the development of a flow cytometry assay, which demonstrated strong expression of TRPA1 in human lung myofibroblasts, human airway smooth muscle cells but not lung mast cells. The most cited anti-TRPA1 antibodies lack sensitivity and/or specificity for hTRPA1. We have identified two anti-TRPA1 antibodies which detect hTRPA1 specifically. Previously published data regarding human TRPA1 protein expression may need revisiting.

scholarly journals Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

2002 ◽  
Vol 364 (3) ◽  
pp. 641-648 ◽  
Author(s):  
Hwei Ling ONG ◽  
Jinglong CHEN ◽  
Tim CHATAWAY ◽  
Helen BRERETON ◽  
Lei ZHANG ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Western Blot ◽  
Airway Smooth Muscle ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Airway Smooth Muscle Cells ◽  
Transient Receptor

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the α- and β-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92kDa or 120kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated β-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

scholarly journals Zinc-dependent lysosomal enlargement in TRPML1-deficient cells involves MTF-1 transcription factor and ZnT4 (Slc30a4) transporter

2013 ◽  
Vol 451 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Ira Kukic ◽  
Jeffrey K. Lee ◽  
Jessica Coblentz ◽  
Shannon L. Kelleher ◽  
Kirill Kiselyov
Keyword(s):  
Transcription Factor ◽  
Transient Receptor Potential ◽  
Regulatory Element ◽  
Transcriptional Response ◽  
Receptor Potential ◽  
Published Data ◽  
Zinc Metabolism ◽  
High Concentrations ◽  
Transient Receptor ◽  
Zinc Levels

Zinc is critical for a multitude of cellular processes, including gene expression, secretion and enzymatic activities. Cellular zinc is controlled by zinc-chelating proteins and by zinc transporters. The recent identification of zinc permeability of the lysosomal ion channel TRPML1 (transient receptor potential mucolipin 1), and the evidence of abnormal zinc levels in cells deficient in TRPML1, suggested a role for TRPML1 in zinc transport. In the present study we provide new evidence for such a role and identify additional cellular components responsible for it. In agreement with the previously published data, an acute siRNA (small interfering RNA)-driven TRPML1 KD (knockdown) leads to the build-up of large cytoplasmic vesicles positive for LysoTracker™ and zinc staining, when cells are exposed to high concentrations of zinc. We now show that lysosomal enlargement and zinc build-up in TRPML1-KD cells exposed to zinc are ameliorated by KD of the zinc-sensitive transcription factor MTF-1 (metal-regulatory-element-binding transcription factor-1) or the zinc transporter ZnT4. TRPML1 KD is associated with a build-up of cytoplasmic zinc and with enhanced transcriptional response of mRNA for MT2a (metallothionein 2a). TRPML1 KD did not suppress lysosomal secretion, but it did delay zinc leak from the lysosomes into the cytoplasm. These results underscore a role for TRPML1 in zinc metabolism. Furthermore, they suggest that TRPML1 works in concert with ZnT4 to regulate zinc translocation between the cytoplasm and lysosomes.

scholarly journals Optimized Flow Cytometric Detection of Transient Receptor Potential Vanilloid-1 (TRPV1) in Human Hematological Malignancies

2021 ◽  
Author(s):  
Sofia Omari ◽  
Dominic Geraghty ◽  
Alhossain Khalafallah ◽  
Pooja Venkatc ◽  
Yvette Shegog ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Transient Receptor Potential ◽  
Mononuclear Cells ◽  
Receptor Potential ◽  
Clinical Samples ◽  
Acute Monocytic Leukemia ◽  
Transient Receptor Potential Vanilloid ◽  
Transient Receptor

The ectopic overexpression of transient receptor potential vanilloid-1 (TRPV1) has been detected in numerous solid cancers including breast, prostate, pancreatic, and tongue epithelium cancer. However, the expression of TRPV1 in hematological malignancies remains unknown. Here we show through in silico analysis that elevated TRPV1 mRNA expression occurs in a range of hematological malignancies and present an optimized flow cytometry method to rapidly assess TRPV1 protein expression for both cell lines and primary patient samples. Three anti-TRPV1 antibodies were evaluated for intracellular TRPV1 detection using flow cytometry resulting in an optimized protocol for the evaluation of TRPV1 in hematological malignant cell lines and patients peripheral blood mononuclear cells (PBMC). Overexpression of TRPV1 was observed in THP-1 (acute monocytic leukemia) and U266B1 (multiple myeloma, MM), but not U937 (histiocytic lymphoma) compared to healthy PBMC. TRPV1 was also detected in all 49 patients (including B-cell non-Hodgkins lymphoma (B-NHL), MM and others, and 20 healthy controls. TRPV1 expression was increased in 8% of patients (MM=2, B-NHL=2). In conclusion, we provide an optimized flow cytometry method for routine expression analysis of clinical samples and show that TRPV1 is increased in 8% of patients with hematological malignancies.

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