Anti fatty acid synthase antibody

The lysis buffer was RIPA buffer with protease inhibitor cocktail. The cells were added to lysis buffer and then underwent an alternant vortex and stored at 4 °C for half an hour. Then the cell lysis was centrifuged at 16,000 × g for 20 min at 4 °C. The Laemmli buffer was used to denature the samples. TGX™ FastCast™ Acrylamide Kit (Bio-rad), APS, and TEMED were used to make the gels. The PVDF membrane was blocked by 5% BSA solution for 1 h. Primary (first) antibodies included Caspase-3 antibody (Cell Signaling Technology, #9662), anti-fatty acid synthase antibody (Abcam, ab22759), Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology, #9101S), and anti-Bcl-xL antibody [E18] (Abcam, ab32370). The membrane was incubated with the primary antibody overnight at 4 °C, rinsed with TBST, and then incubated with the second antibody for 1 h at room temperature. ECL Prime western blotting reagents (GE Healthcare) were used to develop. The developer was myECL Imager (Thermo Fisher Scientific).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine calf serum (BCS), penicillin and streptomycin mixture were obtained from Gibco (Grand Island, NY, USA). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and insulin were purchased from Sigma Aldrich (St Louis, MO, USA). Free fatty acid, adiponectin, and leptin Enzyme-linked immunosorbent assay (ELISA) kits were purchased from YiFeiXue Biotechnology (Nanjing, China) and Jian-Cheng Biotechnology (Nanjing, China). GLUT4 rabbit antibody, anti-PTEN antibody, anti-Fatty Acid Synthase antibody and Wortmannin were purchased from Abcam Biotechnology (Cambridge, UK). Total Akt rabbit polyclonal antibody and p-Akt-ser473 rabbit polyclonal antibody were supplied by Santa Cruze (California, USA). Goat anti-rabbit IgG (H + L) dylight 488 was supplied by Bioworld (Nanjing, China). Total PI3K rabbit polyclonal antibody, p-PI3K rabbit polyclonal antibody, total IRS1 rabbit polyclonal antibody, p-IRS1-ser307 rabbit polyclonal antibody, Na–K-ATPase rabbit antibody, β-actin rabbit polyclonal Antibody, and goat anti-rabbit IgG antibody were all provided by Nanjing Enjing Biotechnology (Nanjing, China).

Total protein was extracted from liver and fat using RIPA buffer supplemented with protease inhibitor cocktail (cOmplete from Millipore-Sigma) with a tissue homogenizer (2X 30 s at 6000 rpm). Then samples were incubated with gentle agitation for 30 min at 4 degrees, followed by centrifugation for 20 min at 13 000 rpm. 5 ug protein lysates were run on 4–15% Criterion TGX Precast Gel (Bio-Rad, Denmark). Proteins were transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare, UK). After blocking (5% BSA at room temperature for one hour), the membranes were incubated overnight at 4 °C with the following primary antibodies: Anti-Acetyl-Coenzyme Carboxylase antibody (EP687Y) (ab45174), and Anti-Fatty Acid Synthase antibody (ab22759), both 1:4000 dilution from Abcam. The membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (Dako, P0448 diluted 1:4000) and afterward, the antigen–antibody complex was visualized using an enhanced chemiluminescence system (ECL, Amersham ECL Plus, GE Healthcare). All Western blots were normalized to total protein, measured using Stain-Free technology, a method previously validated by others^{35 (link),36 (link)}. The total protein gel image can be found in Supplementary information S1, Fig. 2.