Anti pt172 ampkα antibody

Total proteins were extracted using extraction buffer (50 mM Tris‐Base, 150 mM NaCl, 10 mM NaF, 10 mM Na3Vo4, 1× protease inhibitor cocktail, and 0.2% (v/v) Triton X‐100). After centrifugation at 16 000 g for 10 min at 4°C, the supernatants were collected into new tubes. After protein concentrations were measured by the Bio‐Rad protein assay, the total proteins in the supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride membranes. For immunoblotting, primary antibodies such as anti‐MYC (Roche), anti‐HA (Roche), anti‐GFP (Clontech, Mountain View, CA, USA), anti‐GST (Cell signaling, Danvers, MA, USA), anti‐SnRK1.1 (Agrisera, Vannas, Sweden), anti‐pT172‐AMPKα antibody (Cell Signaling), and anti‐Actin11 (Agrisera) were used (1 : 1000). Infrared‐800‐conjugated secondary antibody was added (1 : 10 000). The signal was detected using an IR‐image detector Odyssey (Li‐Cor, Lincoln, NE, USA), and quantitative analysis was carried out with ImageJ.

Total proteins were extracted from plants or protoplasts using extraction buffer (50 mM Tris-Base, 150 mM NaCl, 10 mM NaF, 10 mM Na₃Vo₄, 1x protease inhibitor cocktail, and 0.2% [v/v] Triton X-100). After the cell lysate was centrifuged, the total proteins in the supernatants were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. For immunoblotting, the primary antibodies anti-GFP (Abcam, Cambridge, UK); anti-pT172-AMPKα antibody (Cell Signaling Technology, Danvers, MA, USA); and anti-Actin (Agrisera, Vännäs, Sweden) were used (1:1000), and then an HRP-conjugated secondary antibody (Abcam, Cambridge, UK) was added (1:10,000). The signal was detected using a Fusion SL (Vilber Lourmat, Paris, France). All experiments were performed at least three times with similar results. Representative protein blot data are shown.