Ap1063

Protein lysates were generated from ≈80 mg of finely cut adipose tissue by homogenization in lysis buffer 28 using a tissue lyzer (Qiagen, Germany). Immediately after homogenization, SDS was added to a concentration of 2%. Samples were then centrifuged at 16,000g for 20 min at 4°C to generate lysates. The bicinchoninic acid assay (Thermo Fischer Scientific, MA) was used to determine the protein concentration in the samples and samples were adjusted to a protein concentration of 0.5 µg/µl in sample buffer.
An equal amount of protein was separated by SDS‐polyacrylamide gel electrophoresis (SDS PAGE) and blotted on to a PVDF membrane by semidry blotting. The membrane was then blocked for 1 h in 3% fish gel (FG) (Sigma Aldrich, Copenhagen, Denmark) and incubated overnight in primary antibody against AMPKα1 protein (Hardie G.), AMPK^Thr172 phosphorylation (#2535s Cell Signaling Technologies (CST), Danvers, MA), HSL protein, and HSL^Ser660 and ^Ser565 as well as perilipin (#8334S CST), GLUT4 protein (#PAI‐1065, ABR), PEPCK protein (#10004943, Cayman Chemical Co., Ann Arbor, MI), PDK4 protein (Hardie G.), PDH‐E1α protein (Hardie G.), PDH^Ser300 phosphorylation (Hardie G.), and PDH^Ser232 phosphorylation (#AP1063, Millipore). The following day the membrane was incubated in secondary antibodies (Dako) in 3% FG and quantified using LAS 4000 and Image Quant TL 8.1 (GE Healthcare, Germany).