Rat heart tissues and NRVCs were lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor cocktail. Cell lysate was centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatants were used as samples. Equal amounts of protein (30 μg) were separated in sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore, Billerica, MA, USA). After blocking with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated at 4 °C overnight with a primary antibody against β1adrenergic receptor (β1-AR, diluted 1:1000; Abcam), α-MHC (diluted 1:2000; Proteintech), β-MHC (diluted 1:600; Proteintech), STIM1 (diluted 1:1000; Proteintech), Orai1 (diluted 1:1000; Abclonal, Cambridge, MA, USA), TRPC6 (diluted 1:1000; Proteintech), or GAPDH (diluted 1:5000; Affinity Biosciences). GAPDH was used as the internal loading control. The membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Affinity Biosciences) at 1:5000 dilution for 40 min at room temperature. Bands were visualized with ECL-Plus reagent (Millipore), and the band density was quantified using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA).
Trpc6
Manufactured by Proteintech
Sourced in United States
TRPC6 is a membrane protein that functions as a calcium-permeable cation channel. It is involved in the regulation of cellular calcium homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Cal51 cells, by Leibniz Institute DSMZ (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Affinity Biosciences (1 mentions)
β mhc, by Proteintech (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ecl plus reagent, by Merck Group (1 mentions)
Stim1, by Proteintech (1 mentions)
Orai1, by ABclonal (1 mentions)
β1 ar, by Abcam (1 mentions)
Gel pro analyzer, by Media Cybernetics (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Paclitaxel, by Selleck Chemicals (1 mentions)
Myc y69, by Abcam (1 mentions)
Bi 749327, by MedChemExpress (1 mentions)
Esrp1, by Thermo Fisher Scientific (1 mentions)
Nfat inhibitor, by Cayman Chemical (1 mentions)
Cd44 fitc im7, by BioLegend (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Hcc1806, by American Type Culture Collection (1 mentions)
2 protocols using trpc6
1
Western Blot Analysis of Cardiac Proteins
2
Immunofluorescence and Immunoblotting Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HMLER cells were provided by R. Weinberg (Massachusetts Institute of Technology). MDA-MB-231 and HCC1806 cells were purchased from the American Type Culture Collection, and the TE3 variant of MDA-MB-231 cells was provided by S. Tavazoie (Rockefeller University). CAL-51 cells were obtained from the Leibniz Institute DSMZ, Germany. The TRPC6 inhibitor BI-749327 was purchased from MedChem Express (Catalog no. HY-111925). The paclitaxel was purchased from Selleckchem (Catalog no. S1150). The NFAT inhibitor was purchased from Cayman Chemicals (Catalog no. 13855). The following Abs that were used for flow cytometry were purchased from BioLegend: CD24–allophycocyanin (APC; ML5, BioLegend), CD44-FITC (IM7, BioLegend) and anti-rabbit FITC (MRM-47, BioLegend), anti-Rabbit-555 (Invitrogen-A32732), TRPC6 (18236–1-AP, Proteintech). For immunoblotting, the following Abs were used: Tubulin [Cell Signaling Technology (3873)], Beta-Actin (3700S, Cell Signaling Technology)TRPC6 (ACC-017, Alomone Labs), GAPDH (14C10) [Cell Signaling Technology 2118S], α6A (1A10) (MAB1356, Millipore), and α6B (6B4) (MAB1358, Millipore), Myc (Y69) (32072, Abcam), ESRP1 (PA5–25833, Thermofisher), HA (3724, Cell Signaling Technology), and GST-HRP (5475, Cell Signaling Technology). For immunofluorescence the following antibodies were used: TRPC6 (18236–1-AP, Proteintech), TAZ (clone M2–616, 560235, BD-Pharmingen).
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