Anti beta actin

Proteins were extracted using a buffer containing 25 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na₃VO₄, and protease inhibitor cocktail (Roche Korea, Seoul, Korea). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK, and anti-beta-actin (AbFrontier, Seoul, Korea). Membranes were then probed with the anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Antibody–antigen complexes were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Korea). For the quantification of phospho-AMPK and total-AMPK, ATTO image analysis software (CS analyzer 4, Tokyo, Japan) was used.

Proteins were extracted using the lysis buffer containing 25 mM hydroxyethylpiperazine ethane sulfonic acid (HEPES), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 10 mM NaF, 2 mM Na VO, and a protease inhibitor cocktail (Roche Korea, Seoul, Republic of Korea). Proteins were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany) using a wet transfer system. Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-total AMPK, anti-phosphorylated AMPK, anti-total Akt, anti-phosphorylated Akt (Cell Signaling Technology, Beverly, MA, USA), PTP-MEG2 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-beta-actin (AbFrontier, Seoul, Republic of Korea). Membranes were then probed with an anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Antibody–antigen complexes were detected using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Republic of Korea).