In vitro GST pull-down assay of AsJAZ1 and AsB2510.

(A) Purification of GST-AsB2510 expressed in E. coli Rosetta (DE3). M: protein marker; lane 1: total protein from E. coli Rosetta (DE3) containing empty pGEX-6p-1; lane 2: total protein from E. coli Rosetta (DE3) containing GST-AsB2510; lane 3: GST-AsB2510 purified using glutathione Sepharose 4B column. (B) Purification of His-AsJAZ1 expressed in E. coli Rosetta (DE3). M: protein marker; lane 1: total protein from E. coli Rosetta (DE3) expressing His-AsJAZ1; lane 2 and lane 3: His-AsJAZ1 purified by nickel-agarose column. (C) SDS-PAGE and western blotting of the samples obtained by GST pull-down. His-AsJAZ1 incubated with either the immobilized GST tag or the beads alone served as the negative control samples. Anti-His antibody was applied for the western blot. AsJAZ1-NH, AsJAZ1 with an N-terminal His tag. Blank, purified His-AsJAZ1.