Anti total ampkα

Total protein was extracted from frozen hearts, resolved and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by standard Western blotting procedures [7] (link). A total of 20 µg protein was fractionated using NuPAGE 4–12% Bis-Tris (Invitrogen) gels and transferred to a Protran nitrocellulose transfer membrane (Whatman). The antibodies used for Western blotting were as follows: anti-CaMKKβ (H-95) (Santa Cruz Biotechnology, Dallas, TX, USA), 1∶500; anti-total-CaMKI (sc-33165) (Santa Cruz Biotechnology), 1∶1000; anti-phospho-CaMKIV (sc-28443-R) (Santa Cruz Biotechnology), 1∶500; anti-total-CaMKIV (sc-55501) (Santa Cruz Biotechnology), 1∶1000; anti-phospho-AMPKα (Cell Signaling Technology, Beverly, MA, USA), 1∶500; anti-total-AMPKα (Cell Signaling Technology), 1∶1000; and anti-GAPDH (Cell Signaling Technology), 1∶2000. Anti-phospho-CaMKI (Thr177) was a kind gift from Dr. Naohito Nozaki (Kanagawa Dental College, Yokosuka, Kanagawa, Japan) and used at the dilution of 1∶50. Anti-rabbit IgG (GE Healthcare) and anti-mouse IgG (GE Healthcare) were used as secondary antibodies at a dilution of 1∶2000.

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).