C ebpα antibody

Chromatin immunoprecipitation (ChIP) assays were performed by using EZ-ChIP Kit-17-371 (Millipore) following a previously described method (Deng et al. 2016 (link)). Precleared chromatin was incubated with the C/EBPα antibody (Abcam) or normal rabbit IgG (Abcam) antibody overnight at 4°C. Purified DNA from the samples and the input controls were analyzed for the presence of ACOX1 promoter sequences containing putative C/EBPα response elements using qRT-PCR. The primers used here are listed in Supplementary Table 5.

Protein was extracted from cultured cells by RIPA lysis buffer (Yeasen, Shanghai) with phenylmethylsulfonyl fluoride (PMSF) (1:100; Beyotime, China) and quantified with the BCA Protein Assay Kit (Beyotime). Protein lysates were separated on 15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (0.22 μm; Millipore, Danvers, MA, USA). Then western blotting was carried out. In brief, the membranes were blocked with 5% milk at room temperature for 1 h, incubated with primary antibody overnight at 4 °C, and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000; Yeasen, Shanghai) for 1 h at room temperature. Proteins were detected by ECL reagent (Millipore). The results were obtained using a Tanon imager (Tanon, Shanghai, China). The primary antibodies used were as follows: PPARγ antibody (1:1000; Abcam), AP2 antibody (1:1000; Abcam), LPL antibody (1:1000; Abcam), CEBPα antibody (1:1000; Abcam), DSP antibody (25318-1-AP, 1:500; Proteintech, Wuhan, China), β-catenin antibody (51067-2-AP, 1:1000; Proteintech), GAPDH antibody (10494-1-AP, 1:10,000; Proteintech), and HRP-labeled ACTB antibody (1:10,000; Proteintech).