Phospho ampkα thr172 40h9 rabbit mab

Tetraethylorthosilicate (TEOS), Na₂CO₃, ethanol, methanol, ammoniumhydroxide (NH₃·H₂O), hexadecyl-trimethyl-ammoniumbromide (CTAB) and potassium permanganate (KMnO₄) were purchased from SinopharmChemReagent Co., Ltd. (China). Metformin, lipopoly-saccharide (LPS) and Recombinant Murine IFN-γ were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Murine IL-4 was provided by PeproTech Biotechnology Co., Ltd. (Suzhou, China). Polyclonal antibodies CD47, CD80 and CD206 were obtained from Proteintech Group, Inc. (Wuhan, China). Coumarin-6 was ordered from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Mouse Tumor Necrosis Factor Alpha and Mouse IL-10 ELISA kit was provided by ABclonal Biotechnology Co., Ltd. (Wuhan, China). ELISA kits of Mouse iNOS and Mouse Arg-1 were obtained from Jonln Biotechnology Co., Ltd. (Shanghai, China). FITC-Anti-Mouse CD80 Antibody, FITC-Anti-Mouse CD206 and APC-Anti-Mouse CD206 Antibody were provided by Elabscience Biotechnology Co., Ltd. (Wuhan, China). β-Actin, AMPKα (D63G4) Rabbit mAb and Phospho-AMPKα (Thr172) (40H9) Rabbit mAb were ordered from Cell Signaling Technology, Inc. (MA, USA). DSPE-PEG-M2pep was purchased from SunLipo NanoTech Co., Ltd. (Shanghai, China).

Metformin hydrochloride (D150959) was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in sterile saline. Compound C (6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine) was obtained from Tocris (Cat. No. 3093) and dissolved in dimethyl sulfoxide and further diluted with sterile saline. Metformin and Compound C (10 mg/kg) were injected intraperitoneally. Phospho-Acetyl-CoA Carboxylase (Ser79) (3661, 1:500), Acetyl-CoA Carboxylase (C83B10) rabbit mAb (3676, 1:1000), AMPKα (D63G4) rabbit mAb (5832, 1:1000) and Phospho-AMPKα (Thr172) (40H9) rabbit mAb (2535, 1:1000) were obtained from Cell signaling technology (Beverly, MA, USA). The mouse monoclonal antibody β-actin (A1978, 1:5000) was the product of Sigma-Aldrich (St Louis, MO, USA). HRP-conjugated secondary antibodies for western blot were diluted as 1: 10,000, and were from Thermo Fisher Scientific (Waltham, MA, USA).

Cells were transferred into treatment solutions 2 h prior to lysis. The lysis procedure consisted of three washes in chilled PBS before final aspiration and addition of lysis buffer. The lysis was performed rapidly to prevent activation of AMPK. Cell lysate was centrifuged at 13,500 rpm at 4 °C for 20 min. The protein concentration of the supernatant was determined by Bradford assay and diluted to 0.6 mg/mL in 10% Wes Sample Buffer (042-195, ProteinSimple, San Jose, CA, USA) and run according to manufactures instructions on a WES capillary electrophoresis western blot machine (Protein Simple). Primary antibodies AMPKβ 1/2 (57C12), rabbit mAb (#4150, Cell Signalling, Beverly, MA, USA), phospho-AMPKα (Thr172) (40H9), rabbit mAb (#2535, Cell Signalling, Beverly, MA, USA), and phospho-acetyl-CoA carboxylase (Ser79) antibody (#3661, Cell Signalling, Beverly, MA, USA) were diluted in WES antibody dilution buffer at 1 in 150, 1 in 150 and 1 in 200, respectively. Peak areas were obtained from the Compass software (Protein Simple V2.5.11) at 30 s exposure. Data from at least two independent samples were used to calculate ratios of peak areas.