For tissue distribution of mRNA and protein expressions, data on the target genes were obtained from “The Tissue Atlas” category of “The Human Protein Atlas/HPA” (http://www.proteinatlas.org/ ) [51 (link)]. The mRNA expression from the “The Genotype-Tissue Expression/GTEx Dataset” was chosen for demonstration with reference to the normalized consensus dataset Immunohistochemistry staining was performed on normal human tissue samples. AR expression was detected with a rabbit antibody (1:250, HPA004733, Sigma-Aldrich) and validated with another rabbit antibody (1:500, GTX62599, GeneTex). ACE2 expression was primarily detected with a rabbit antibody (1:250, HPA000288, Sigma-Aldrich) and confirmed with a mouse antibody (1:5000, CAB026174, R&D Systems); TMPRSS2 expression was detected with a rabbit antibody (1:300, HPA035787, Sigma-Aldrich). The information on expression intensity and specific cell types that express respective genes were extracted from the staining reports for each staining type in the database.
Hpa035787
Manufactured by Merck Group
HPA035787 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis. The core function of this product is to perform specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
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4 protocols using hpa035787
1
Tissue Expression Profiling of AR, ACE2, and TMPRSS2
2
TMPRSS2 and SARS-CoV-2 Spike Protein
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Cells were lysed in TXNE buffer (1% Triton X‐100, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, protease inhibitors) for 30 min on ice. Equal amounts (20–50 μg) of cell lysates were analyzed by Western blot. The following antibodies were diluted in WB buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): rabbit anti‐human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), rabbit anti‐human actin (Sigma cat#A2066, 1:2,000), and human anti‐S serum derived from a convalescent individual (1:1,000). Species‐specific secondary DyLight‐coupled antibodies were used (diluted 1:10,000 in WB buffer) and proteins were revealed using a Licor Imager. Images were processed using Image Studio Lite software.
3
SNAP-ACE2 Protein Expression Analysis
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Lysates from cells transfected with SNAP-ACE2 were resolved in SDS-PAGE gel (10%), followed by protein transfer to nitrocellulose membranes. Membranes were blocked in 5% non-fat dried milk in TBS (10 mM Tris-HCl, pH 8, 150 mM NaCl), and immunoblotted with primary antibodies against the FLAG tag (1:1,000; F7425, Sigma-Aldrich), or against TMPRSS2 (1:1,000; HPA035787; Sigma-Aldrich) diluted in 0.3% BSA in TBS (overnight, 4°C). Immunoreactivity was revealed using secondary antibodies coupled to 680 or 800 nm fluorophores (1:15,000, diluted in 0.3% non-fat dried milk in TBST; LI-COR Biosciences, Lincoln, NE, USA), and readings were performed with the Odyssey LI-COR IR fluorescent scanner (LI-COR Biosciences).
4
Western Blot Analysis of ACE2 and TMPRSS2
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Cells were lysed in TXNE buffer (1% Triton X‐100, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, protease inhibitors) for 30 min on ice. Equal amounts (20–50 μg) of cell lysates were analyzed by Western blot. The following antibodies were diluted in WB‐buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): goat anti‐human ACE2 (R&D cat#AF933, 1:2,000), mouse anti‐human ACE2 (Adipogen AC18F cat #AG‐20A0032‐C100, 1:1,000), rabbit anti‐human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1,000), mouse anti‐Flag tag (Sigma cat# F1804, 1:1,000), rabbit anti‐human actin (Sigma cat#A2066, 1:2,000), and mouse ascite anti‐SARS S, (1:1,000; Siu et al, 2008 ). Specie‐specific secondary DyLight‐coupled antibodies were used (diluted 1:10,000 in WB‐buffer) and proteins revealed using a Licor Imager. Images were quantified and processed using Image Studio Lite software.
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