Anti phosphorylated ampkα

Cultured preadipocytes were isolated using ice-cold M-PER (Thermo Fisher Scientific, USA), protease inhibitor (Sigma-Aldrich, USA), and 2 mM Na₃VO₄ (Thermo Fisher Scientific, USA). Homogenated cells were then mixed with an equal volume of 2×standard sodium dodecyl sulfate (SDS) sample loading buffer (Invitrogen, Waltham, MA, USA). Gradient gels were used for SDS-polyacrylamide gel electrophoresis separation of proteins. Membranes were then incubated overnight at 4°C in primary antibodies: anti-AMPKα, rabbit polyclonal (Cell signaling, Danvers, MA, USA) with dilution of 1:1,000, anti-phosphorylated AMPKα, rabbit polyclonal (Cell signaling, USA) with dilution of 1:1,000. Membranes were then incubated with a secondary antibody, Alexa-Fluor 633, goat anti-rabbit, dilution at 1:2,000 dilution for 2 h at the room temperature. After three 10 min washes, membranes were visualized using enhanced chemiluminescent substrate, Western blotting reagents (Thermo Fisher Scientific, USA), and exposure to film (MR, Kodak, Rochester, NY, USA). Density of the bands were quantified using Imager Scanner II and Image Quant TL software. To reduce the variation between blots, tissue lysates of both groups were run in a single gel. Band density was normalized according to the glyceraldehyde 3-phosphate dehydrogenase (Cell signaling, USA) content within each sample.

Metformin (1,1-dimethylbiguanide hydrochloride), phenformin (N-[2-phenylethyl]imidodicarbonimidic diamide monohydrochloride), and biotin were purchased from Sigma. Anti-HMGB1, anti-phosphorylated p38, anti-total p38, anti-phosphorylated AMPKα, and anti-total AMPKα antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-His antibody was from Sigma. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Anti-HMGB1 neutralizing monoclonal antibody and control anti-keyhole limpet hemocyanin (KLH) antibody (IgG2a isotype control) were generated as described (40 (link)). LPS was from InvivoGen (San Diego, CA). TAK-242 was from EMD chemicals (San Diego, CA). Other chemicals were purchased from Sigma or Wako (Osaka, Japan).