Clarity western ecl chemiluminescent

Frozen heart tissue samples were homogenized in liquid nitrogen using a mortar and pestle. Protein lysate was extracted from tissues using 1X RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, and 1% sodium deoxycholate), and from cells using 1X lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Tween-20). Conventional immunoblotting procedures were used to determine the levels of selected proteins involved in DDR signaling and the circadian clock. The following antibodies were used: Actin (Santa Cruz Biotechnology; Cat #s sc-1616), Bmal1 (Bethyl Laboratories; Cat #s A302-616A), c-Caspase 3, pH2a.x, H2a, and Rev-erbα (Cell Signaling Technology; Cat #s 9664P, 9718S, 12349S and 13418S respectively). The appropriate anti-mouse, anti-rabbit, and anti-goat HRP-conjugated secondary antibodies (Sigma-Aldrich) were used for detection with Clarity Western ECL chemiluminescent (Bio-Rad Laboratories) and/or SuperSignal West Femto (Thermo Fisher Scientific) reagent methods with Bio-Rad ChemiDoc XRS+ imager.