Immunoblot Analysis of STIM and Orai Proteins

Ebv-miR-BART13-3p transfected and mock-transfected HSG cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA buffer (Promega, WI, USA) supplemented with Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics, IN, USA). Lysates were then centrifuged at 12,000 × g for 30 min at 4 °C. Twenty micrograms of protein was loaded and resolved in a 4%–12% NuPAGE gels (Invitrogen, CA, USA). Anti-STIM1 (Cell Signaling Technology Cat# 5668S, RRID:AB_10828699), anti-Orai1 (Sigma-Aldrich Cat# AV50118, RRID:AB_1848716), anti-STIM2 (Cell Signaling Technology Cat# 4917S, RRID:AB_2198021) anti-β-actin (Cell Signaling Technology Cat# 3700P, RRID:AB_10828322), and Anti-TRPC1 antibody (Willoughby et al., 2014 (link)) were used at 1:1000, 1:1000, 1:1000, and 1:400 dilution, respectively. Protein bands were detected by chemiluminescence and exposed to X-ray film (Kodak, New York).