Ampkβ2

IP procedures were performed as previously described [22] (link). Briefly, exogenous myc-AMPKα1 was immunoprecipitated using anti-myc-tag antibody (9B11, Cell Signaling Technology), exogenous R6 was immunoprecipitated using anti-FLAGtag antibody (F3165, Sigma) and endogenous AMPK was immunoprecipitated using a combination of anti-AMPKα1 and anti-AMPKα2 antibodies raised in sheep (kindly provided by Dr D.G. Hardie, University of Dundee, Dundee, UK), followed by incubation with Protein G-Sepharose beads (GE Healthcare). Western blot analysis was carried out using primary antibodies against the following: myc-tag, total AMPKα, AMPKβ1, AMPKβ2, phospho-AMPK-Thr-172 (all from Cell Signaling Technology), FLAG-tag (F3165, Sigma), and phospho-AMPKβ2-Thr-148 [22] (link). Detection was performed according to its primary antibody using anti-rabbit (Cell Signaling Technology) and anti-mouse (Dako) horse radish peroxidase (HRP)-conjugated secondary antibodies, followed by chemiluminescence.
In order to investigate the role of glycogen, myc-AMPKα1 was immunoprecipitated using the anti-myc-tag antibody after the addition of the glycogen mimic β-cyclodextrin (β-CD; 5 mM, Sigma) for 1 h at 4 • C. Subsequently, immune complexes were electrophoresed by SDS/PAGE and analysed by Western blot analysis, as described above.