Peroxidase coupled goat anti rabbit igg

Cell lysates (30 μg protein each sample) dissolved in Laemmli buffer 5×, boiled for 5 min were resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels (SDS-PAGE, Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis they were transferred to polyvinylidene fluoride membranes (PVDF, Bio-Rad Laboratories), which were incubated overnight at 4 °C with specific primary antibodies: anti phospho-Akt (p-Akt; 1:1000; Ser473, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti Akt (1:1000; Santa Cruz Biotechnology), anti UCP1 (1:1000; 4E5, Santa Cruz Biotechnology), anti p-AMPKα/β (1:1000; D-6, Santa Cruz Biotechnology), AMPKα/β (1:1000; Santa Cruz Biotechnology), anti Mfn2 (1:1000; H-68, Santa Cruz Biotechnology). The membranes were washed and then incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma), peroxidase-coupled rabbit anti-goat IgG and horseradish peroxidase-coupled goat anti-mouse IgG (Sigma) for 45 min and were developed through a nonradioactive method using Western Lightning Chemiluminescence (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA). Phosphorylated protein expression was calculated as a ratio towards specific total protein or β-actin (1:5000; C4, Santa Cruz Biotechnology).

Total seedling or inflorescence protein samples were extracted from 100 mg of ground tissue with NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) according to Ref. 69 (link). Equal volumes of extract from different samples were separated on precast 4–20% Criterion gradient gels (Bio-Rad) before transfer to nitrocellulose membranes (Amersham Biosciences Protran Premium 0.45 μm, GE Healthcare) using a trans-blot blotting apparatus (Bio-Rad). Primary antibodies were detected using peroxidase-coupled goat anti-rabbit IgG (Sigma) and visualized using chemiluminescence SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).