Cd36 nb400 144

To extract proteins, hearts were washed with cold PBS homogenized, and then cocktails of RIPA, protease, and phosphatase inhibitors (ratio 100:1:1) were added. Protein concentrations were standardized before being electrophoresed on 10–12% gels and transferred to a 0.45 m PVDF membrane (Millipore Immobilon®-P; IPVH08100). Membranes were blocked in TBST with 2% BSA, and proteins of interest were blotted with the antibodies listed here: Glut1 (E4S6I) (Rabbit mAb #73015; CD36 (NB400-144) Novus Biologicals; 1:2000); Cleavage Caspase 3 (Asp175) (D3E9); Cell Signaling Technology; #9579 Rabit mAb;1:1000), CD73 (Abcam; Ab175396; 1:1000), anti-GATA4 (Abcam; ab84593; 1:1000), TNAP (ALPL) Rabbit mAb (A0514); PAP (PSAP) Rabbit mAb (A21195); GAPDH (Proteintech; 10494–1-AP; 1:1000) and HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech; SA00001-2; 1:1000). Enhanced chemiluminescence (Tanon, China) was used to visualize the membranes.

Isolation of total and nuclear extracts was performed as described elsewhere [21 (link)]. Proteins (30 μg) were separated by SDS-PAGE on 10% (w/v) acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Merck Millipore). Western blot analysis was performed using antibodies against HIF1α (sc-10,790), LIPIN1 (sc-98,450), Histone H3 (sc-10,809), SREBP1 (sc-365,513), Nrf2 (sc-722), NQO1 (sc-393,736), PPARγ (sc-7273) (Santa Cruz Biotechnology), SIRT3 (#5490), phospho-mTOR Ser2481 (#2974), mTOR (#2972), Keap1 (#4678 s) (Cell Signaling Technology Inc., Danvers, MA), GAPDH (MAB374) (Merck Millipore), CD36 (NB400–144) (Novus Biologicals, Centennial, CO), VLDLR (AF2258) (R&D Systems, Minneapolis, MN). Detection was performed with the Western Lightning™® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad, Hercules, CA).