Immunoblot analysis of CMV coat protein

Immunoblot analysis of CMV CP extracted from virus-infected plants was performed according to the method described previously (Du et al., 2014a (link)). Briefly, upper systemically-infected leaves were harvested at 14 dpi and used for extraction of total soluble proteins, which were separated in a 15% SDS–PAGE gel, and transferred onto a nitrocellulose membranes. Membranes were incubated with the polyclonal anti-CP serum (Agadia). For analyzing GFP protein, total soluble protein was extracted from leaves expressing GFP or GFP-2b fusion proteins at 5 dpi. Primary antibody binding on membranes was detected using horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz) and ECL substrate (Thermo-Fisher).