Immunoblotting Analysis of Cardiac Proteins

Proteins were extracted from heart tissues with K150T buffer (150 mM KCl, 50 mM Tris-HCl pH7.4, 0.125% Na deoxycholate, 0.375%Triton X-100, 0.15% NP-40, 4mM EDTA, 50 mM NaF) and immunoblotting was performed as previously described [3 (link)]. In brief, equal amount of proteins (15 ug) were resolved on 4-12% NuPAGE Bis-Tris gel and transferred to PVDF membrane. Pierce Reversible Protein Stain Kit was used to detect total proteins for loading normalization. Primary antibodies used in immunoblotting were phospho-S6(Ser235/236), S6, phospho-AKT (S473), phospho-AKT (T308), phospho-ULK (S757), ULK, phospho-AMPK (T172), AMPK, phospho-GSK-3β (S9), GSK-3β, Atg5 (all from Cell Signaling), phospho S657-PKCα, PKCα (both from Santa Cruz), LC3 (from Novus Biologicals), TFAM (mtTFA; from Abcam). Secondary antibodies used were donkey anti-rabbit IgG secondary antibody and goat anti-mouse IgG secondary antibody (both from Thermo Scientific. AlphaView Software was used for image acquisition and signal quantification.