PKG activity assay and Western blot analysis

Cardiac PKG activity was measured with the CycLex Cyclic GMP-dependent protein kinase (cGK/PKG) Assay Kit (Cyclex, MBL International Corp, Nagoya, Japan) from tissue homogenates. Activity was measured according to the manufacturer’s instructions. Spectrophotometic absorbance was measured at 450 nm. Results were normalized as per mg of protein, and relative activity is presented.

Homogenates of whole hearts were used for Western blotting. Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane. Non-specific binding was blocked with 5% skim milk in 1X Tris-buffer containing 0.1% Tween-20 for an hour. Blots were incubated overnight with primary antibodies (1:000, Cell Signaling Technology, MA, USA) and visualized with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000, Vector Laboratories, Burlingame, CA, USA) for 1 hour. Blots were developed with ECL chemi-luminescence detection reagent (Bio-Rad, CA, USA).