Phospho ampkalpha thr172 antibody

Five thoraces were added to 75 µl of lysis buffer (62 mM Tris pH7.5, 0.1% SDS with protease and phosphatase inhibitors). The samples were boiled for 5 min at 95 °C and centrifuged to collect the supernatant. Sample loading buffer was added, and the samples were then electrophoresed on 10% Mini-PROTEAN TGX precast protein gels (Bio-Rad). The protein bands were transferred to the nitrocellulose membrane (Bio-Rad) and incubated with AMPK-alpha antibody (Cat.: ab80039, 1:3000, Abcam), Phospho-AMPK-alpha (Thr172) antibody (Cat.: 2535 S, 1:1000, Cell Signaling), alpha Tubulin antibody (Cat.: ab52866, 1:3000, Abcam). For the secondary antibody, horseradish peroxidase-conjugated Goat anti-rabbit IgG (ab6721, Abcam) and Rabbit anti-mouse IgG (ab6728, Abcam) were used at 1:10,000 dilution. Clarity Max or Clarity Western ECL substrate (Bio-Rad) was used to detect the signal, and the corresponding band was quantified using ImageJ software.

Western blotting techniques were modified from previous methods [29 (link), 59 (link)]. Proteins tagged with the HA epitope (Snf1, Glc7) were detected with Anti-HA probe (Santa Cruz) diluted 1:2,000. Goat anti-mouse IRDye 800CW (Li-Cor) diluted 1:5,000 was used as the secondary antibody. For detection of phosphorylated Snf1, Phospho-AMPKalpha (Thr172) antibody (Cell Signaling) diluted 1:1,000 was used. Goat anti-rabbit IRDye 800CW (Li-Cor) was used as the secondary antibody at a 1:10,000 dilution. Proteins tagged with the V5 epitope (Hxk2, Reg1) were detected with the Anti-V5 probe (Invitrogen) diluted 1:1,000. Goat anti-mouse IRDye 680 (Thermo) diluted 1:10,000 was used as the secondary antibody. Blots were visualized using an Odyssey Infrared Imager (Li-Cor), and band quantification was performed using Odyssey software.