Phosphor ampk thr172

Liver proteins were extracted using radioimmunoprecipitation buffer (Cell Signaling Technology, Beverly, MA, USA). Total protein containing 40 μg was separated on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Bio-Rad, CA, USA). After blocking, the primary antibodies employed were: β-actin (Biovision, Mountain View, USA), adenosine monophosphate (AMP)-activated protein kinase α [(AMPK) Cell Signaling Technology], phosphor-AMPK (Thr172) [(p-AMPK) Cell Signaling Technology], sterol regulatory element binding protein 1 [(SREBP1) Novus Biologicals, Littleton, CO, USA] and sterol regulatory element binding protein 2 [(SREBP2) Proteintech, Manchester, UK]. The membranes were incubated overnight at 4°C with primary antibodies, followed by incubation with horseradish peroxidase-linked secondary antibodies for 1 h at room temperature. The blots were incubated with appropriate horseradish peroxidase-linked secondary antibodies and then detected by enhanced chemiluminescence (Western Lightning ECL Pro; PerkinElmer, Waltham, MA, USA). Signal intensities were quantitated using the Quantity One Software (BioRad, Hercules, CA, USA).