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.