Secondary antibodies conjugated to hrp

Total proteins were extracted from BLCA tissue samples or cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) containing protease inhibitors. The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). Proteins (30 μg) were separated on 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h and then incubated overnight at 4 °C with primary antibodies against NUSAP1 and TGFBR1 (cat. nos. ab137230 and ab31013, respectively; 1:1000 dilution for each; Abcam); GAPDH (cat. no. AB0037, 1:5000 dilution; Abways); E-cadherin, vimentin, N-cadherin, CDK1, cyclin B1, Smad2/3, P-Smad2/3, Bax, Bcl2 and cleaved-caspase3 (cat. nos. 3195T, 5741T, 13116T, 9116T, 12231T, 8685T, 8828s, 5023T, 3498T and 9964T, respectively; 1: 1000 dilution for each; Cell Signaling Technology). After washing with TBST three times, the membranes were incubated with secondary antibodies conjugated to HRP (Cell Signaling Technology, USA) for 1 h at room temperature. After washing with TBST three times, the bands were visualized using an ECL system (Bio-Rad Laboratories). The relative protein expression was analyzed using Quantity One software (Bio-Rad).

To measure the phosphorylation state of cTnI and phospholamban (PLB), isolated cardiac myocytes were plated in 35‐mm dishes, infected with adenovirus, and cultured for 40 hours. After 40 hours, myocytes were pretreated with timolol (2 μmol/L) and, in some cases, also with lepB (18.5 nmol/L) for 30 minutes followed by PE (20 μmol/L) for 5 minutes at 37°C. Whole‐cell lysates were prepared as described previously.^{16 (link)} Western blots were performed with primary antibodies to Thr144 phospho‐cTnI (1:1000; Abcam, Cambridge, MA), Ser23,24 phospho‐cTnI, total cTnI (1:1000; Cell Signaling), Thr17 phospho‐PLB (1:5000; Badrilla Ltd., Leeds, UK), Ser16 phospho‐PLB, and total PLB (1:2000; Millipore, Billerica, MA) followed by appropriate secondary antibodies conjugated to HRP (1:5000, Cell Signaling). All antibodies were prepared in 5% BSA in TBS‐T (20 mmol/L of Tris‐HCl, 150 mmol/L of NaCl, pH 7.6, 0.1% Tween‐20). Blots were developed using enhanced chemiluminescence (GE Healthcare, Fairfield, CT) and analyzed using ImageJ software (National Institutes of Health [NIH], Bethesda, MD).