Ab133585

We analysed the expression of cardiac lipid metabolism proteins by the Western blot method as previously described in detail.²⁸ As severe LV hypertrophy is related to the impaired angiogenesis pathway, we also studied the expression of key proteins that regulate angiogenesis and signal hypoxia. Primary antibodies: HIF1α (ab463, Abcam; 1:500); fatty acid transporter (CD36; ab133625, Abcam; 1:1000); carnitine palmitoyltransferase 1 (CPT1; NBP1‐59576, Novus Biological; 1:1000); fatty acid‐binding protein (FABP3; ab133585, Abcam; 1:1000); acyl‐CoA dehydrogenase (ACADL; ab196655, Abcam; 1:3000); medium‐chain acyl‐CoA dehydrogenase (MCAD; ab110296, Abcam; 1:1000); 2,4‐dienoyl‐CoA reductase (DECR1; ab198848, Abcam; 1:2000). AMP‐activated protein kinase (AMPK; 2532 s, Cell Signaling; 1:1000); phosphorylated AMPK (Thr 172) (pAMPK; 2535 s, Cell Signaling; 1:500); SIRT1 (8469 s, Cell Signaling; 1:1000); PPARα (ab8934, Abcam; 1:1000); retinoid X receptor (RXRα; 3085 s, Cell; 1:1000); peroxisome proliferator‐activated receptor gamma coactivator 1α (PGC1α; 20,658‐1‐AP, Proteintech; 1:1000). Targeted bands were normalized to the expression of cardiac GAPDH (sc‐32,233, Santa Cruz Biotechnology; 1:2000).

To verify some of the differentially expressed proteins via 2-DE and shotgun proteomics, we conducted Western blot analysis for two proteins involved in lipid metabolism, as platelet glycoprotein 4 (Cd36) and fatty acid-binding protein (Fabp3). The following primary antibodies were used: anti-Cardiac Fabp (1:1000; ab133585, Abcam, Cambridge, MA, USA), and anti-CD36 (1:1000; ab133625). For full experimental details of this analysis, see supplementary materials and methods.