Western blot

Differentiated C2C12 cells were serum-starved for 48h in low-glucose Gibco DMEM and then stimulated with ANP, BNP, or CNP at 200nM for 30min, or subjected to a 30min treatment with 200nM 8-Br-cGMP as a positive control to induce downstream PKG activity and 200nM 8-Br-cAMP to act as a negative control. Cells were lysed and sonicated in lysis buffer (25 mM HEPES at pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM glycerophosphate, 0.9% Triton X-100, 0.1% IGEPAL, 5 mM sodium pyrophosphate, 10% glycerol, 1x Complete protease inhibitor and 1x PhoSTOP phosphatase inhibitor cocktail). For Western blotting, 40 μg of protein was resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad), incubated overnight at 4°C with primary antibodies, and followed by inoculation with alkaline phosphatase–conjugated secondary antibody for 1 hour at room temperature. Blots were incubated with Amersham ECF substrate (Cytiva, RPN5785) and images were acquired by Bio-Rad digital ChemiDoc MP with IR (VUMC Molecular Cell Biology Resource).