Western Blot Analysis of Cellular Proteins

Immunoblotting was performed as previously described [21 (link)]. Cells were prepared by RIPA (radio-immunoprecipitation assay) buffer with phosphatase inhibitors (Sigma). After vortex 30 s, cells were lysed at ice for 15 min and centrifuged at 12000 rpm for 15 min at 4°C. Supernatant was denatured and used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad; Hercules, CA, USA). Subsequently, the membranes were immunoblotted with the corresponding primary antibodies followed by peroxidase-conjugated secondary antibodies. The bonds were visualized by Chemiluminescence (Thermo Scientific; Waltham, MA, USA). SPOP (1:1000 Proteintech; 16750-1-AP), Flag (1:1000 Proteintech; 66008-2-Ig), GAPDH (1:2000 Cell Signaling Technology; #5174), FH (1:1000 Proteintech; 10966-1-AP), ELOVL2 (1:500 Abcam; EPR11880), ACADL (1:1000 Proteintech; 17526-1-AP) and Vinculin (1:2000 Sigma; V4505) antibodies were used in the study.