Anti mybpc3

Protein concentration in heart tissue lysates was measured using the bicinchoninic acid method (Thermo Scientific, 23227, Waltham, MA, USA). Equivalent amounts of each protein extract were loaded into each well, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. Blots were probed with the following antibodies: anti-βMHC (Abcam, Cambridge, UK, ab50967), anti-MyBPC3 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-137237), anti-PPARα (Abcam, ab34509), anti-PPARβ/δ (Cell Signaling, Danvers, MA, USA, 2443S), anti-CD36 (Novus Biologicals, Minneapolis, MN, USA, NB400-144), anti-CPT1α (Abcam, ab1285568), anti-GLUT4 (Abcam, ab33780), anti-Akt (Cell Signaling, 9272S), anti-pS473 Akt (Cell Signaling, 9271S), anti-GSK3β (Cell Signaling, 5676S), anti-pSer9 GSK3β (Cell Signaling Technology, 9336S), and GAPDH (Proteintech, HRP-60004). Signals were visualized using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Image Lab Software version 6.0.1 (Bio-Rad) was used to quantify the differences in the fold induction of protein expression normalized to that of GAPDH.

Dulbecco’s Modified Eagle Medium was from Cellgro. Fetal bovine serum was from Sigma-Aldrich. Restriction enzymes were from New England Biolabs. Protease inhibitor cocktail was from EMD Millipore Corporation (Cat#: 539131) and phosphatase inhibitor cocktail was from Cell Signaling Technology (Cat#: 5870). Horseradish peroxidase (HRP)-conjugated anti-Flag antibody was from Sigma-Aldrich (Cat#: A8592). Rabbit anti-MYPT1/2 was from Abcam (Cat#: ab32519). Anti-PPP1CB and anti-Phospho-MLC-2v (Ser15) antibodies were from Thermo Fisher Scientific (Cat#: PA5-28225 and PA5-104265, respectively). Anti-PPP1CC antibody was from Origene (Cat#: TA308937). Anti-Phospho-MYPT1 (Thr696) antibody was from Cell Signaling Technology (Cat#: 5163). Anti-MYBPC3 (Cat#: sc-137237) and HRP-conjugated anti-β-actin antibody (Cat#: sc-47778-HRP) were from Santa Cruz Biotechnology. Peroxidase-conjugated Goat anti-Rabbit IgG was from Jackson ImmunoResearch (Cat#: 111-035-003). SuperSignal West Pico Plus was from Pierce (Cat#: 34580). IRDye 680RD Goat anti-mouse and IR DYE 800CW Goat anti-Rabbit and Intercept Blocking Buffer were from LI-COR. Other general chemicals and supplies were from Thermo Fisher Scientific or Sigma-Aldrich.