Rabbit pab

Western blot analysis was carried out using antibodies targeting phospho-(Y416)-Src Rabbit pAb (Cell Signaling Technology, Danvers, MA, USA, Cat# 2101, RRID:AB_331697), Src (L4A1) Mouse mAb (Cell Signaling Technology Cat# 2110, RRID:AB_10691385), and PARP (46D11) Rabbit mAb (Cell Signaling Technology Cat# 9532, RRID:AB_659884). Mouse monoclonal antibodies were used in conjunction with anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology Cat# 7076, RRID:AB_330924). Rabbit polyclonal antibodies were used in conjunction with anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology Cat# 7074, RRID:AB_2099233). Equal loading was assessed using vinculin (Abcam Cat# ab129002, RRID:AB_11144129) or β-actin (Cell Signaling Technology Cat# 3700, RRID:AB_2242334) targeting antibodies.

A lysis buffer containing 50 mM Tris–HCl at pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1 mM PMSF, and protease inhibitor cocktail (10 mg/mL leupeptin, 10 mg/mL pepstatin A, and 10 mg/mL aprotinin) was used to lyse the hBMSCs on ice for 30 min for Western blot analysis. The protein parts were collected by centrifugation under the condition of 15,000 g at 4°C for 10 min, subjected to 10% SDS–PAGE, and finally transferred onto nitrocellulose membranes. The transferred membranes were blocked by 5% BSA and cultured with specific antibodies at 4°C overnight. An enhanced chemiluminescence detection system (#WBKLS0500, Millipore, Billerica, MA, United States) was used in accordance with the manufacturer’s instructions for visualization after adding the horseradish peroxidase–labeled secondary antibody. The primary antibodies used were as follows: Runx2 mouse mAb (1:1000, #ab76956, Abcam, United States), Smad1 Rabbit mAb (1:1000, #6944, Cell Signaling Technology, United States), phospho-Smad1/5/8 Rabbit pAb (1:1000, #4086, Cell Signaling Technology, United States), and GAPDH Rabbit mAb (1:1000, # 2118, Cell Signaling Technology, United States).

The heart was homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 50 mM β-glycerophosphate, 30 mM NaF, 2 mM EDTA, 2 mM EGTA, 30 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100) containing protease inhibitors (cOmplete^TM, Roche). The homogenate was centrifuged at 1000 g to pellet the nucleus and debris. The heat-dissociated proteins (40 µg/lane) were fractionated by SDS-PAGE (Mini-PROTEAN® TGX, Bio-Rad) and detected with Western blot by chemiluminescence (SuperSignal^TM West Pico PLUS, Thermo Fisher Scientific). Blots developed on the chemiluminescence film (Amersham Hyperfilm ECL; GE Healthcare) were quantified using ImageJ software (NIH). The antibodies used for Western blot are as follows:Y1135-phosphorylated IGF-1 receptor β (pIGF1Rβ; Rabbit mAb; DA7A8; Cell Signaling Technology), IGF-1 receptor-β (IGF1Rβ; Rabbit pAb; 3027; Cell Signaling Technology), Y694-phosphorylated signal transducer and activator of transcription 5 (pSTAT5; Rabbit mAb; C11C5; Cell Signaling Technology), total STAT5 (Rabbit mAb; D206Y; Cell Signaling Technology), cleaved caspase 3 (Rabbit mAb; 9664; Cell Signaling Technology), total caspase 3 (Rabbit mAb; 8610; Cell Signaling Technology), and β-actin (HRP-conjugated Rabbit mAb; 3683; Cell Signaling Technology).