Western blot analysis

After specific drug treatments, cells were incubated in lysis buffer (PMSF:RIPA, 1:100; Sigma-Aldrich; Merck KGaA) for 20 min on ice. After insoluble debris was pelleted by centrifugation at 12,000 x g for 15 min at 4˚C, the supernatants were collected and protein concentrations were assessed by the bicinchoninic acid method. After boiling the samples for 10 min, the protein samples (10 µg/lane) were fractionated by SDS-PAGE (10-12% polyacrylamide gels), transferred to polyvinylidene fluoride membranes (EMD Millipore) and blocked with 5% non-fat milk for 2 h at room temperature. The membranes were then incubated overnight at 4˚C using appropriate dilutions of primary antibodies against CaSR (1:1,000; cat. no.; Abcam), proliferating cell nuclear antigen (PCNA, 1:250; cat. no. BM0104; Boster Biological Technology), smooth muscle actin α (α-SMA; 1:500; cat. no. BM0002; Boster Biological Technology, Wuhan, China), calponin (1:400; cat. no. BM4088; Boster Biological Technology), osteopontin (OPN; 1:1,000; cat. no. ab8448; Abcam), renin (1:250; cat. no. bs-6184R; BIOSS), AT1R (1:1,000; cat. no. ab9391; Abcam, UK), Bcl-2 (1:200; cat. no. BA0412; Boster Biological Technology), Bax (1:200; cat. no. BA0315; Boster Biological Technology) and β-actin (1:1,000; cat. no. TA-09; ZSGB-Bio; OriGene Technologies, Inc.). Membranes were then washed three times with Tris-buffered saline containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:20,000; Boster Biological Technology) for 2 h at room temperature. Detection was performed with an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.). Band intensity was quantified using Bio-Rad Quantity One software (version 4.3.0; Bio-Rad Laboratories, Inc.) with β-actin as an internal control.