Protein Extraction and Western Blot Analysis

Agroinfiltrated N. benthamiana leaves were harvested and homogenized in liquid nitrogen, followed by addition of equal volumes of gel loading buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 200 mM β-mercaptoethanol, 0.2% bromophenol blue). After vortex mixing and boiling, the samples were centrifuged for 10 min at 12000 g. Proteins remaining in the supernatant were resolved by 12.5% SDS-PAGE, followed by staining with Coomassie brilliant blue or were transferred to nitrocellulose filters for Western blot analysis [85 (link)].
Western blots were performed as described previously [67 (link), 84 (link)]. Briefly, nitrocellulose membranes containing transferred proteins (Hybond-C, GE Healthcare) were blocked, incubated with primary antibodies raised against the γb, Actin, GFP, FLAG or HIS proteins. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase or protein A-alkaline phosphatase (Sigma-Aldrich), and the signals were detected with an enhanced chemiluminescence (ECL) detection kit (GE Healthcare) or by color reactions developed by incubating with substrate solution (0.33 mg/mL NBT and 0.165 mg/mL BCIP in 100 mM Tris-HCl buffer, pH 9.5, containing 100 mM NaCl). The results were recorded with a Cannon scanner.