Anti foxo1

Lysing porcine pre-adipocyte and extracting the total protein by radioimmunoprecipitation assay (RIPA) lysis buffer (Vazyme, Nanjing, China) following the protocol. The protein is quantified by the BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Loading the protein onto the 12% SDS-PAGE, every protein sample is 15μg/well. After one-hour electrophoresis, the protein sample is transferred to the polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, USA). Blocking the membrane with 5% BSA (Bovine serum albumin), overnight incubation with primary antibody at 4℃, subsequently. After membrane washing the appropriate secondary antibody is used to incubate. The ECL Chemiluminescence Detection Kit (Thermo Scientific, USA) is used to detect and images are captured by the VersaDoc 4000 MP system (Bio-Rad, USA). The antibodies respectively are anti-C/EBPα (ZEN BIO, 383901), anti-FoxO1 (ZEN BIO, 383312), anti-AdipoQ (ZEN BIO, 383390).

For co-IP, the pre-adipocytes is washed thrice by PBS, then using the radioimmunoprecipitation assay (RIPA) lysis buffer (Vazyme, Nanjing, China) that contains 1% phenylmethylsulfonyl fluoride (PMSF) to lyse the cell, whole cell lysate is incubating with antibodies conjugated Protein A/G PLUS-Agarose (Santa Cruz, sc-2003) in RIPA at 4°C overnight. The antibodies respectively are anti-C/EBPα (ZEN BIO, 383901) and anti-FoxO1 (ZEN BIO, 383312). After the overnight incubation, washing the immunoprecipitates thrice with the fresh RIPA before subjection to immunoblotting (IB).

Using the ChIP Assay Kit (Boytime, Nanjing, China) following the manufacturer's instruction to detect ChIP-PCR. In brief, using 1% formaldehyde to cross-link porcine pre-adipocyte at 37℃ for 10 minutes. 1×glycine is used to quench the cross-linked porcine pre-adipocyte at room temperature for 5 minutes. Using VCX750, the ultrasonic cell smash machine (Sonics, United States) to smash porcine pre-adipocyte and obtain DNA fragments. Subsequently, following the rule that per 100ml cell lysis buffer is incubating with 1 mg of anti-C/EBPα (ZEN BIO, 383901) and anti-FoxO1 (ZEN BIO, 383312) at 4℃ overnight, 60ml Protein A Agarose/SalmonSperm DNA is used to isolate immunoprecipitated complexes at 4℃ for 4 hours, washing the complexes as following: low salt wash buffer, high salt wash buffer, LiCl wash buffer once, and TE buffer twice. Final ChIP DNA fragments are subjected to PCR analysis using an AdipoQ promoter specific primers, as shown at Supplementary Table S1.