Cd36 18836 1 ap

Total protein from cell lines was extracted using Radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail. Protein concentrations were determined by a BCA protein assay kit (P0009; Beyotime, Shanghai, China). Equal amounts of protein were separated via denaturing Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred electrophoretically onto polyvinylidene difluoride (PVDF) membranes and blocked, and then incubated overnight at 4°C with primary antibodies directed against HMGB3 (ab75782; Abcam), CD36 (18836-1-AP; Proteintech, Manchester, UK), E-cadherin (14472 S, CST), Snail (3879 S, cell signaling technology [CST]), vimentin (SK2482712C, Invitrogen), and β-actin. Corresponding horseradish peroxidase-conjugated secondary antibody was subsequently added for 2 hours at room temperature, and protein bands were visualized by exposure to film. The relative protein levels were calculated based on β-actin as the loading control.

HEK293T cells were seeded in 6-well plates 16 h before transfection. The indicated expression vectors (Myc-CDK9, HA-HDAC1, or HA-HDAC2: 1 μg) were co-transfected with or without the Flag-METTL3 expression vector (1 μg) in HEK293T cells. For the Co-IP of HDAC1/2 and METTL3, total cell lysates were first incubated with DNase1 (200 U/ml) at 37 °C for 30 min to digest genomic DNA. These lysates were then immunoprecipitated with Myc, Flag, or HA beads at 4 °C for 2 h. Immunoblotting was performed using the indicated antibodies. Antibody dilutions were as follows: METTL3 (96391, Cell Signaling Technology), 1:2500; Lamin B1 (12987-1-AP, Proteintech), 1:5000; Tubulin (sc-5286, Santa Cruz), 1:5000; β-actin (60008-1-Ig, Proteintech), 1:5000; CDK9 (11705-1-AP, Proteintech), 1:2500; p-CDK9 (2549, Cell Signaling Technology), 1:2500; CD36 (18836-1-AP, Proteintech), 1:5000; Flag (F1804, Sigma), 1:5000; Myc (16286-1-AP, Proteintech), 1:5000; HDAC1 (5356, Cell Signaling Technology), 1:2500; HDAC2 (5113, Cell Signaling Technology), 1:2500; and Caspase3 (9662, Cell Signaling Technology), 1:2500. Other reagents were listed in Supplementary Table 3.