Anti acc1

Anti-IRS-1, anti-pIRS-1 (S307), anti-AKT (T308), anti-AKT, anti-p-mTOR (S2448), anti-mTOR, anti-p-p70S6K (T389), anti-p70S6K, anti-p-S6 (S235/S236), anti-S6, anti-p-p38MAPK (T180/Y182), anti-p38MAPK, anti-p-ERK1/2 (T202/Y204), anti-ERK1/2, anti-p-SAPK/JNK (T183/Y185), anti-SAPK/JNK, anti-p-AMPK (T172), anti-AMPK, anti-ACC1, and anti-FASN monospecific antibodies were purchased from Cell Signaling (Danvers, MA). Insulin, Oil Red O, and anti-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Anti-SREBP1 antibody was bought from Becton-Dickinson (Franklin Lakes, NJ). Anti-SREBP2 and anti-ChREBP antibodies were bought from R&D Systems (Minneapolis, MN). Combined protease and phosphatase inhibitor cocktails (Halt™) and Tissue-Tek™ optimum cutting temperature (OCT) compound were bought from Thermo Fisher Scientific (Hampton, NH). Forward and reverse oligonucleotide primers (Additional file 1: Table S1) to carry out quantitative PCR analysis were designed according to a published program (OligoArchitectTM online, Sigma-Aldrich, St. Louis, MO). DNA primers were synthesized by Integrated DNA Technologies (Coralville, IA).

The following antibodies and working concentrations were used: anti-Actin C11 (1:5000, Sigma Aldrich A2066, for western blot), anti-SREBP1 2A4 (1:500, Santa Cruz Biotechnology sc13551, for western blot), anti-SREBP1 H160 (1:100, Santa Cruz Biotechnology sc8984, for immunofluorescence), anti-SREBP2 (1:500, BD Bioscience 557037, for western blot), anti-SCD1 (1:1000, Abcam ab19862, for western blot), anti-GAPDH (6C5) (1:5000, Santa Cruz Biotechnology sc32233, for western blot), anti-AMPK (1:1000, Cell Signalling 2532S, for western blot), anti-AMPK phospho Thr172 (1:1000, Cell Signalling 2531S, for western blot), anti-ACC1 (1:1000, Cell Signalling 3676S, for western blot), anti-ACC1 phospho Ser79 (1:1000, (Cell Signalling 11818S, for western blot), anti-Farnesyl (1:1000, AB4073 Merck Millipore), anti-Hsp90 (1:2000, Santa Cruz Biotechnology sc13119, for western blot), anti-MLC2 (1:1000, Cell Signalling 3675S, for western blot), anti-MLC2 phospho Ser19 (1:500, Cell Signalling 3671S, for western blot and immunofluorescence), anti-FAK C-20 (1:1000, Santa Cruz Biotechnology sc-558, for western blot) and anti-FAK phospho Y397 (1:1000, Abcam ab81298, for western blot).

BBR and BRB (≥98%) were purchased from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China). Metformin (MET) (≥98%) and fenofibrate (FEN) (≥98%) were obtained from Shanghai Aladdin Biochemical Technology Co. Ltd and the National Institute for Food and Drug Control, respectively. The mouse insulin ELISA kit was purchased from ZCIBIO Technology Co. Ltd (Shanghai, China). The total cholesterol (TC), triglyceride (TG), high-density lipid-cholesterol (HDL-c), low-density lipid-cholesterol (LDL-c), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), and creatinine (CRE) kits were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Anti–GLUT2 antibody, the primary antibody used for Western blotting, was obtained from Abcam plc (Cambridge, United States). Anti-ACC1, anti-FAS, anti-GSK3β, and anti–p-GSK3β-Ser9 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, United States). Anti-ATGL was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, United States). Anti-GK, anti-PPARα, anti-CPT1, anti-CD36, anti-PPARγ, anti-PEPCK, anti-G6Pase, and anti–β-actin were purchased from Proteintech Group, Inc. (Wuhan, China). A secondary antibody was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd (Beijing, China).

Protein expression was assessed by immunoblot analysis of cell lysates (20–60 μg) in RIPA buffer in the presence of the following antibodies: including anti-p-AMPK (Affinity, AF3423), anti-AMPK (Affinity, AF6423), anti-p-ACC1 (Cell Signaling Technology, 11818s), anti-ACC1(Cell Signaling Technology, 4190s), anti-ACOX1 (Santa Cruz Biotechnology, sc-517306), anti-p-mTOR (Affinity, AF3308), anti-mTOR (Affinity, AF6308), anti-SGLT2 (Abcam, ab37296), anti-LC3B (Cell Signaling Technology, 12741S), anti-Beclin1 (Cell Signaling Technology, 4122s), anti-p62/SQSTM1 (Proteintech, 18420-1-AP), and anti-GAPDH (Abcam, ab8245).
Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States). Dulbecco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS, Gibco) were obtained from Gibco; palmitate (PA) was obtained from Sigma-Aldrich (P9767); compound C (Comp C) was purchased from AbMole (M2238), chloroquine (CQ, S4157) and dapagliflozin (S1548) were bought from Selleck; and Oil Red O and triglyceride detection kit (G1262; BC0625) were obtained from Solarbio.

After transfection, cells were treated in a lysis buffer containing a complete protease inhibitor cocktail (Shanghai Roche Pharmaceuticals), followed by the extraction and quantification of proteins. Subsequently, the protein was transferred the into nitrocellulose membranes, blocked, and incubated in specific primary antibody at 4 °C overnight. The membranes were washed and incubated with second antibody for 2 h at room temperature. Finally, the protein bands were visualized using chemiluminescence detection system. The following antibodies were used: anti- FASN (Abcam, UK), anti- ACC1 (Cell Signaling Technology, Danvers, MA, USA), anti-FABP5 (Cell Signaling Technology, Danvers, MA, USA); anti-CD36 (Santa CruzBiotechnology, Santa Cruz, CA, USA).