Mouse anti flag antibody

Total proteins were harvested from plant samples as described previously [49 (link)]. Proteins were isolated on 15% SDS polyacrylamide gels and then transferred onto polyvinylidene fluoride membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blotted with a mouse anti-flag antibody (1:5000; Abmart) or rabbit anti-BrYV movement protein antiserum (1:1000) and then incubated with goat anti-mouse HRP antibody (1:3000; Bio-Rad, Hercules, CA, USA) or goat anti-rabbit HRP antibody (1:3000; Sigma-Aldrich, St. Louis, MO, USA), respectively. Finally, the membranes were detected using an enhanced chemiluminescence detection method.

Immunoblotting analysis was conducted as previously described [54 (link)]. The proteins were denatured and separated using 12% SDS-PAGE, and transferred to a PVDF membrane (Millipore, IPVH00010). The membrane was blocked with 5% skim milk powder at 25°C for 1 h, and then probed with a mouse anti-GST antibody (Abmart, M20007), mouse anti-HIS antibody (Abmart, M30111), mouse anti-FLAG antibody (Abmart, M20008), mouse anti-ubiquitin monoclonal antibody (Sigma-Aldrich, U0508), mouse anti-MYC antibody (Abmart, M20002), or mouse anti-actin monoclonal antibody (Sigma-Aldrich, A0480). At last, the membrane was probed a goat anti-mouse HRP-linked antibody (Abmart, M21001), and were analysed using the FDbio-Dura ECL kit (FD8020, Hangzhou, China).