Immunoblot analysis
CsJAZ6 protein fused to the Myc tag and CsEGL3 and CsTTG1 proteins fused to the Flag tag were prepared for co-immunoprecipitation assays. Transiently transfected tobacco leaves expressing either CsEGL3-Flag or CsTTG1-Flag with CsJAZ6-Myc were lysed with a buffer. The buffer contained 50 mM Tris, 50 mM NaCl, 1 mM EDTA 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 mM MG132 (Sigma-Aldrich), and protease inhibitor cocktail (Roche). Lysates were added the Anti-Flag Magnetic Beads (Sigma-Aldrich) and incubated for 4 h at 4 °C. Then, the beads were washed three times, then solubilized sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min. Samples were subsequently analyzed by 4%-12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with anti-Myc peroxidase antibody. ECL Plus Western Blotting kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect the bound antibody.