Ab68191

Salts and organic solvents used in solution preparations were purchased from Fisher Scientific (Leicestershire, UK), Sigma Chemicals (USA), or Merck Darmstadt (Germany), with the highest grade of purity commercially available. Antibodies used were targeted to AMPK and (Thr172)AMPK (#2532, #2535, Cell Signaling, USA); F4/80, GLUT2, ACC, (Ser79)ACC, and (Tyr1163)IRβ (ab74383, ab54460, ab72046, ab68191, and Ab60946, Abcam, UK); IRβ (sc-57342, Santa Cruz Biotechnology, USA) argpyrimidine (AGE06B, Nordic MUbio, Netherlands); and MG-H1 (HM5017, Hycult Biotech, Netherlands). Calnexin was used as loading control (AB0037, SICGEN, Portugal).

Proteins were prepared from liver tissues in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors cocktail (Roche, Switzerland). Then SDS-PAGE was conducted and bands were transferred to PVDF membrane, incubating with primary antibodies against toll-like receptor 4 (TLR4), microtubule-associated protein light chain 3 (LC3), phosphor-IκB and phosphor-ACC (diluted at 1:1000, catalog number: ab45104, ab48394, ab12135 and ab68191, respectively, Abcam, Cambridge, UK), phosphor-NFκB, NFκB, IκB, caspase-3, caspase-8, caspase-9, poly-(ADP-ribose) polymerase (PARP), and phosphor-AMPK (diluted at 1:1000, catalog number: 3033, 8242, 4814, 9665, 4790, 9508, 9532, and 2535, respectively, Cell Signaling Technology, Boston, USA). Corresponding secondary antibodies (diluted at 1:5000, catalog numbers: ZDR-5306 or ZDR-5307, Sino Biological, Beijing, China) were added and bands were visualized with enhanced chemiluminescence reagents (Thermo Fisher Scientific, Waltham, USA). Signals were normalized to that of β-actin (diluted at 1:5000, catalog number: 4967, Cell Signaling Technology, Boston, USA).

We purchased aspirin (V900185), LPS (Escherichia coli 0111:B4), and phosphate-buffered saline (PBS) from Sigma-Aldrich (St. Louis, Missouri, USA). A 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (Beyotime Institute of Biotechnology, Shanghai, China) was used to assess intracellular ROS production, and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and compound C were purchased from Selleck Chemicals (Shanghai, China). Additionally, we purchased rabbit anti-rat collagen type II (COL2; ab34712), MMP-3 (ab13533), MMP-13 (ab39012), IL-1β (ab9722), TNF-α (ab6671), AMPK (ab32047), phospho-AMPK (p-AMPK; ab133448), acetyl-CoA carboxylase (ACC; ab45174), phospho-ACC (p-ACC, ab68191), nitric oxide (NO) synthase (iNOS; ab15323), cyclooxygenase-2 (COX-2; ab15191), and nuclear factor (erythroid-derived 2)-like 2 (Nrf-2; ab137550) antibodies, as well as goat anti-rabbit immunoglobulin G (IgG) heavy+light chain (H&L) Alexa Fluor 488 (ab150077) and Alexa Fluor 647 (ab150079) from Abcam (Cambridge, UK). Anti-β-actin and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime. Aggrecan (bs-1223R), ADAMTS-4 (bs-4191R), and ADAMTS-5 (bs-3573R) were purchased from Bioss (Beijing, China).