PC12 cells were collected and treated with RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing 1% (v/v) phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), 0.3% (v/v) protease inhibitor (Sigma-Aldrich; Merck KGaA) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich; Merck KGaA). Western blots were performed as previously described (22 (link)). Membranes were incubated with primary antibodies against TRPC6 (cat. no. ab62461; 1:1,000; Abcam) and anti-GAPDH (cat. no. 2118; 1:5,000; Cell Signaling Technology, Inc.) overnight at 4˚C. Membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (cat. no. ZB-2301; 1:5,000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 2 h at room temperature, followed by three washes with TBS-Tween-20. Enhanced chemiluminescence (ECL; EMD Millipore) was used to determine protein concentrations according to the manufacturer's protocol. Protein signal was detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.). Relative protein expression was normalized to GAPDH. All experiments were repeated three times. ImageJ v1.43b software (National Institutes of Health) was used for densitometric analysis.