Rabbit anti gapdh antibody

Snap-frozen hearts or isolated cardiomyocytes were lysed in the RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM dithiothreitol, protease and phosphatase inhibitors) or the Urea buffer (20 mM HEPES pH 7.4, 1 M NaCl, 8 M urea, protease and phosphatase inhibitors). Proteins were extracted using pestle homogenization and sonication. Cell lysates were centrifuged at 13,000 rpm for 5 minutes at 4°C, and proteins in the supernatant were separated by SDS-PAGE. Proteins were transferred to a PVDF membrane. Membranes were blocked with nonfat milk. The primary antibodies were rabbit anti-Lamin A/C antibody (Santa Cruz, sc-20681, 1:1000), rabbit anti-cGAS antibody (Cell Signaling, #31659, 1:500), rabbit anti-STING antibody (Cell Signaling, #13647, 1:500), and rabbit anti-GAPDH antibody (ABclonal, AC001, 1:1000); Secondary antibodies: anti-rabbit/mouse IgG (H+L) DyLight 680 and 800 (Cell Signaling, 1:5000). Signals were detected and quantified in the Odyssey CLx Imager (LI-COR). The gel after protein transfer was counter-stained by Coomassie to evaluate the loaded protein amount.