For protein extraction from myocardial tissue block, tissue block was weighed and treated with ice-cold lysis buffer (60 µL per mg of tissue) with protease inhibitor, followed by being homogenized with the homogenizer at low speed (30 sec each cycle with a 1-min break on ice) until complete lysis. The protein lysate was centrifuged at 12,000 rpm at 4 ℃ for 20 min, and the supernatant was collected and stored at −80 ℃ for further use.
BCA Protein Assay Kit was used in determining Protein concentration (Thermo Scientific, USA). The protein extract underwent electrophoresis using 10% SDS/PAGE gels and later transferred onto PVDF membrane (Merck Millipore, Germany). Antibodies including vimentin (Santa Cruz, USA), cardiac troponin T (cTnT, Santa Cruz, USA), connexin 43 (Cell Signaling, USA), c-Kit (Santa Cruz, USA), CD31 (Santa Cruz, USA), α-smooth muscle actin (α-SMA, Abcam, USA), α-sarcomeric actin (Abcam, USA), rabbit anti-p38α (Santa Cruz, USA), mouse anti p-p38 (Santa Cruz, USA), GAPDH (Proteintech, USA) were all utilized for Western blot analyses. Western blot bands were quantitated by Quantity One software (BioRad, USA).
BCA Protein Assay Kit was used in determining Protein concentration (Thermo Scientific, USA). The protein extract underwent electrophoresis using 10% SDS/PAGE gels and later transferred onto PVDF membrane (Merck Millipore, Germany). Antibodies including vimentin (Santa Cruz, USA), cardiac troponin T (cTnT, Santa Cruz, USA), connexin 43 (Cell Signaling, USA), c-Kit (Santa Cruz, USA), CD31 (Santa Cruz, USA), α-smooth muscle actin (α-SMA, Abcam, USA), α-sarcomeric actin (Abcam, USA), rabbit anti-p38α (Santa Cruz, USA), mouse anti p-p38 (Santa Cruz, USA), GAPDH (Proteintech, USA) were all utilized for Western blot analyses. Western blot bands were quantitated by Quantity One software (BioRad, USA).