Western Blot Analysis of Plant Proteins

Total protein was extracted as described [8 (link)] by grinding leaf samples in 0.5 mL glycine buffer [19 (link)] per 1 g of leaves. After a Bradford assay, extracts were normalized to 0.5 mg/mL. For Western blotting, 12.5 μg were used and proteins were separated by gel electrophoresis at 150 V for 60 min and transferred to nitrocellulose membranes. GFP was detected with anti-GFP antibody (Merck Millipore, Darmstadt, Germany) at a 1:4000 dilution. 6HIS3xFlag-tagged NSs was detected with anti-Flag-peroxidase antibody (Sigma-Aldrich, St. Louis, MO, USA) at a 1:10,000 dilution. HA-tagged p19 [15 (link)] was detected using anti-HA antibody with peroxidase (Roche, Basel, Switzerland) at a 1:1000 dilution. HSP70 was used as the loading control and detected using primary anti-HSP70 (Merck Millipore, Darmstadt, Germany) at 1:6000 dilution and secondary antibody (goat anti-rabbit immunoglobulin G, NA934-1; GE Healthcare, Little Chalfont, UK) at a 1:10,000 dilution. TuMV CP was detected with anti-CP (PVAS-134 at 1:10,000 dilution) with secondary antibody NA934-1 at a 1:10,000 dilution. Primary and peroxidase-conjugated antibodies were incubated at 4 °C overnight and secondary antibodies for 30 min at room temperature. Chemiluminescence was measured with Clarity Western ECL Substrate and a ChemiDoc^® MP Imaging System (Bio-Rad, Hercules, CA, USA).