Ir image detector odyssey

The total protein was extracted from the protoplasts using an extraction buffer (50 mM Tris-Base, 150 mM NaCl, 10 mM NaF, 10 mM Na₃Vo₄, 1x Complete, and 0.2% Triton X-100). The proteins were separated in 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. For immunoblotting, the primary antibodies anti-HA (Roche), anti-GFP (Abcam), and anti-ACT (Agrisera) were used (1:1000). Next, an HRP-conjugated secondary antibody (Abcam) was added (1:10,000). The signal was detected using an IR-image detector Odyssey (LI-COR, Lincoln USA).

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.