Proteins were extracted from frozen 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, 4 mM EDTA, 50 mM NaF) and quantified by BCA protein assay (Thermo Scientific). Equal amount of proteins (15 µg) were resolved on 4–12% NuPAGE Bis-Tris gel and transferred to PVDF membrane. A Pierce Reversible Protein Stain Kit was used to detect total proteins for normalization of loading.
Primary antibodies used in immunoblotting were OXPHOS (Abcam ab110413, at 1:500), Troponin I (Cell Signaling Technology #4002, 1:1000), pSer23/24-Troponin I (Cell Signaling Technology #4004, 1:1000), pSer150-Troponin I (ThermoFisher PA5-35410; 1:1000), cMyBP-C (Santa Cruz SC-137237, 1:1000), pSer282-cMyBP-C (ALX-215–057 R050, 1:2000).
Secondary antibodies used were donkey anti-rabbit IgG secondary antibody and goat anti-mouse IgG secondary antibody (both from Thermo Scientific). SuperSignal West Pico Chemiluminescent Substrate was used for detection and AlphaView Software (Protein Simple, San Jose, CA), was used for image acquisition and quantification.
Primary antibodies used in immunoblotting were OXPHOS (Abcam ab110413, at 1:500), Troponin I (Cell Signaling Technology #4002, 1:1000), pSer23/24-Troponin I (Cell Signaling Technology #4004, 1:1000), pSer150-Troponin I (ThermoFisher PA5-35410; 1:1000), cMyBP-C (Santa Cruz SC-137237, 1:1000), pSer282-cMyBP-C (ALX-215–057 R050, 1:2000).
Secondary antibodies used were donkey anti-rabbit IgG secondary antibody and goat anti-mouse IgG secondary antibody (both from Thermo Scientific). SuperSignal West Pico Chemiluminescent Substrate was used for detection and AlphaView Software (Protein Simple, San Jose, CA), was used for image acquisition and quantification.