Sc 366

After treatment with stearate, oleate or linoleate, cells were collected and total proteins were lysed using RIPA lysis buffer (Beyotime, Nanjing, China). The homogenates were combined with equal volumes of SDS sample buffer, and the proteins were separated by electrophoresis on a 5~12% polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20, followed by overnight probing with the following primary antibodies: (1) CD36 (N-15) antibody (sc-5522, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (2) ACACA (T-18) antibody (sc-26817, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3) DGAT1 antibody (ab59034, 1:500, Abcam, Cambridge, MA, USA), (4) SREBP1 (C-20) (sc-366, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (5) PPARγ (T-18) antibody (ab19481, 1:500, Abcam, Cambridge, MA, USA), (6) β-actin (C4) antibody (sc-47778, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin was intended to serve as a loading (internal) control. After washing, membranes were incubated with secondary antibody (ABR, Golden, CO, USA) and conjugated to HRP. The chemiluminescent signal was detected using ECL reagents (Beyotime, Nanjing, China) and bands were quantified by image processing software (Image Pro Plus 6.0, Media Cybernetics, Rockville, MD, USA).

Total protein were extracted from 100 mg frozen liver sample as previously described [26 (link)]. Protein concentrations were determined using Pierce BCA Protein Assay kit (Rockford, IL, USA) according to the manufacturer’s instructions. 40 μg of protein was used for electrophoresis on a 10% or 7.5% SDS-PAGE gel. Western blot analysis for HMGCR (bs6625, Bioworld Technology, USA, diluted 1:1000), SREBP1 (sc-366, santa cruz, USA, diluted 1:200), SREBP2 (sc-5603, santa cruz, USA, diluted 1:200), CYP7A1 (ab79847, Abcam, UK, diluted 1:200), CYP27A1 (bs2192, Bioworld Technology, USA, diluted 1:200), BHMT (15965-1-AP, Proteintech, USA, diluted 1:200), AHCYL1 (10658-3-AP, Proteintech, USA, diluted 1:1000), DNMT1 (24206-1-AP, Proteintech, USA, diluted 1:1000), DNMT3a (bs6587, Bioworld Technology USA, diluted 1:500), H3K27me3 (17–622, Millipore, USA, diluted 1:500) was carried out according to the recommended protocols provided by the manufacturers. The β-actin (AP0060, Bioworld, USA, diluted 1:10,000) was used as loading control in the Western blot analysis. Images were captured by VersaDoc 4000MP system (Bio-Rad, USA) and the band density was analyzed with Quantity One software (Bio-Rad, USA).