Anti cpt1α

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.

Total proteins were extracted from HepG2 cells using protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA). Protein concentrations were measured using a BCA Protein Quantitative Assay Kit. The target proteins were blotted with the following antibodies: anti-phospho-p38 (CST, 4511), anti-p38 (CST, 9212), anti-phospho-ERK (CST, 4376), anti-ERK (CST, 4695), anti-phospho-JNK (CST, 4668), anti-JNK (CST, 9252), anti-phospho-AMPK (CST, 2535), anti-AMPK (CST, Thr172, 2532S), anti-phospho-ACC (CST, Ser79, 11818), anti-ACC (CST, 3662), anti-CPT1α (Abcam, ab128568), and anti-GAPDH (Proteintech, 60004-1-Ig). The appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) (Amersham, Little Chalfont Bucks, UK) were used at a 1 : 5000 dilution. An Alpha Q detection system was used to visualize the bound primary antibodies.

Liver tissue were lysed in RIPA buffer containing a protease inhibitor (Beyotime Institute of Biotechnology, China). 30 μg of total protein were separated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and followed by the transfer of proteins to a PVDF membrane. After blocking with 5% skim milk, membranes were incubated with the appropriate primary antibodies overnight at 4 °C. Primary antibodies including anti-phospho-IRS-1^Ser612(CST), anti-IRS-1(CST), anti-phospho-AKT^Ser473(CST), anti-AKT (CST), anti-Glut-2 (Abclonal), anti-Sirt1(Abcam), anti-PPARα (Abclonal), anti-CPT1α(Abcam), anti-AMPK (Abcam). Blots were incubated with an appropriate horseradish conjugated secondary antibody (Beyotime Institute of Biotechnology, China) at room temperature for 1 h. Antibody bound protein was detected by ECL (Merkmillipore) and quantified by scanning densitometry. Relative protein expression was normalized to Tublin expression.