Western Blot
The apex of the ventricular myocardia were homogenized in a lysis buffer containing phosphatase and proteinase cocktail inhibitor, in the ratio 100:1:1 respectively. Lysates of normalized concentrations and treated with reducing agents were denatured at 100°C for 10 min and separated on SDS-PAGE gel. The protein bands were transferred at 100V for 1 h onto polyvinylidene fluoride (PVDF) membranes and blocked with 1% bovine serum albumin in Tris-buffered saline and Tween 20. The primary antibodies employed in this study are as follows: β1AR (ab3442; Abcam), β2AR (ab182136; Abcam), MEF2 (ab64644; Abcam), GRK5 (ab64943; Abcam) GATA4 (ab84593; Abcam), NFAT (ab25916; Abcam), AC5 (PAC-501AP; FabGennix), AC6 (PAC-601AP; FabGennix), AC7 (PAC-701AP; FabGennix), ANP (sc-515701; Santa Cruz Biotechnology), BNP (sc-271185; Santa Cruz Biotechnology), GRK2 (sc-13143; Santa Cruz Biotechnology), p ERK1/2 (4370; Cell Signaling Technology), ERK1/2 (9102S; Cell Signaling Technology), p NF-κB (3033T; Cell Signaling Technology), NF-κB (8242T; Cell Signaling Technology) and GAPDH (10494-1-AP; Proteintech). Blots imaging was done using an enhanced chemiluminescence (Tanon, Shanghai, China). Protein bands were quantified and normalized with their respective GAPDH expressions.