Co-immunoprecipitation of CD36 in IHH Cells

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.