Western Blotting of AMPK Signaling

At the end of the culture periods, protein extracts from total INS-1E β-cells (4.0 × 10⁶ cells/dish) and human islets (200 islets/well) were harvested in lysis buffer as described [33 (link),54 (link)]. Proteins from total cell extracts (10–20 μg/lane) were separated by 8%–10% SDS-PAGE before transfer onto nitrocellulose membrane. The membrane was then probed overnight at 4 °C with rabbit polyclonal antibodies against AMPKα (62 kDa, the antibody detects both the α1 and α2 isoforms of the catalytic subunit, not the regulatory β or γ subunits); p-AMPKα (thr172) (1:1000 dilutions, Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal antibodies against LKB1 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA); and ACTIN (1:5000, Chemicon-Millipore, Zug, Switzerland). After washing, the membranes were incubated for 1 h at RT with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (1:10000, Amersham Biosciences, Buckinghamshire, UK) according to primary antibodies. Proteins were revealed by chemiluminescence (ECL, Amersham Biosciences, Buckinghamshire, UK), analyzed with the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA), and bands were quantified with Scion Image software (Scion Corporation, Frederick, MD, USA).