Anti phospho ampka thr172

Yeast cells were grown for 48 h in YPD liquid medium and, then, inoculated in fresh YPD (initial O.D._600nm 0.25). Flasks were incubated under shaking (200 rpm) at 30°C and growth was followed till O.D._600nm ~1.0 (after 5 h). Culture samples were collected and total protein homogenates were prepared according to Yaffe and Schatz method [46 (link)] with minor modifications. NaOH and β-mercaptoethanol were added to a final concentration of 0.2 M and 1%, respectively. Tubes were incubated on ice for 2 minutes and, then, trichloroacetic acid was added to a final concentration of 6%. After incubation on ice for 10 minutes, tubes were centrifuged at maximum speed for 5 minutes. Supernatant was discarded and pellets were stored at -20°C until use or promptly resusspended in Laemlli sample buffer to obtain a 10-O.D. suspension. Proteins were separated by electrophoresis (SDS-PAGE, 10% SDS-polyacrylamide gel, 100 V, 2 hours) and electrotransferred to Immobilon-P for 20 min at 18 V in 25 mM Tris, 192 mM glycine and 10% methanol, using a trans-blot semi-dry cell (BioRad). Membranes were treated with anti-phospho-AMPKa Thr172 (Cell Signaling) or anti-HA (Sigma) antibodies. Blots were detected using the chemiluminescence ECL Plus kit (GE Healthcare).