Hrp anti rat igg for hsc70

MEFs were harvested using the Qiagen kit with protease inhibitors. Samples were kept on ice for at least 20 min, after which nonsoluble material was pelleted at 20,000g for 20 min at 4 °C. The supernatant was removed and protein concentrations were estimated using the Bio-Rad Dc Protein assay.
For western blot analysis, 20 μg of the protein lysates were run on 4–12% Bis-Tris SDS–PAGE gels (NuPAGE Novex, Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in 5% milk in TBS-T, and incubated overnight at 4 °C with primary antibodies to FABP4 (Cell Signaling), PLIN1 (Cell Signaling) and HSC70 (Abcam, ab19136) as an internal control. Blots were washed in TBS-T and incubated with secondary antibody (horseradish peroxidase (HRP)-anti-rabbit IgG for FABP4 and PLIN1, HRP-anti-rat IgG for HSC70, GE Healthcare) for 1 h at room temperature. After washing, bands were visualized with enhanced chemiluminescence reagent (SuperSignal West Pico, Pierce) and X-Ray film. Protein density was analysed using the ImageJ software (National Institutes of Health, USA). All primary antibodies used were at 1:1000, and all secondary antibodies were at 1:2500 dilutions.