Ab133625

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).

Triacylglycerols (TG), Total cholesterol (TCH), Low-density lipoprotein cholesterol (LDL-C), High-density lipoprotein cholesterol (HDL-C), free fatty acids (FFA) and total bile acid (TBA) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against liver X receptor-α (LXRα) (ab176323), peroxisome proliferator activated receptor γ (PPARγ) (ab45036), sterol regulatory element binding protein 1c (SREBP-1c) (ab28481), cluster of differentiation 36 (CD36) (ab133625), acyl-CoA carboxylase 1 (ACC1) (ab72046), fatty acid synthase (FAS) (ab15285), low-density lipoprotein receptor (LDLR) (ab30532), diacylglycerol acyltransferase 2 (DGAT2) (ab237613) and Goat Anti-Rabbit IgG H&L (HRP) (ab205718) were purchased from Abcam company(Cambridge, UK).

Co-immunoprecipitation (Co-IP) was conducted following the protocol^{115 (link),117} . Hepatocyte (IHH) cancer cells were cultured at 37 °C for 24 h in DMEM media supplemented with 10% FBS and 1% 1× penicillin/streptomycin. The next day, the media was replaced with new serum-free DMEM media supplemented with recombinant TSP1 (Sigma Aldrich, #ECM002-50UG) for 24 h. The treated cells were collected and lysed. The extract solution was divided into three parts as follows: 10% as input, 45% for immunoprecipitation with anti-IgG antibody (Santa Cruz, #sc-2025), and 45% for immunoprecipitation with anti-CD36 antibody (abcam, #ab133625). 1 µg of the desired antibody was added to the extract solution and incubated overnight at 4 °C in the rotator. Concurrently, the beads (Millipore, #16-157) were blocked by mixing with 5% BSA and incubating overnight at 4 °C with rotation. The next day, the blocked beads were incubated with the lysate-antibody mixture for 4 h at 4 °C with rotation. Bound proteins were analyzed by Western blotting.