Screening of primary transgenic plants expressing C-terminally FLAG-tagged HSP90.5. A, Schematic diagram of C-terminally FLAG-tagged HSP90.5 that was constructed in binary vector pGWB402Ω through gateway cloning system. B, Examination of the specificity of polyclonal rabbit anti-HSP90.5 antibody. Immunoblotting was performed using purified cytosolic HSP90 from S. cerevisiae (yHSP82), Arabidopsis (AtHSP90.2) and human (hHSP90α), and ER-localized HSP90 from Arabidopsis (AtHSP90.7) and canine (dGRP94), with 50 ng loaded of each, and different amounts of purified AtHSP90.5. CBB staining represents SDS-PAGE stained with Coomassie brilliant blue. C, Immunoblotting of total leaf lysate proteins from 3-week-old primary transgenic seedlings. A total of 7.5 μg protein was separated by SDS-PAGE and immunoblotted with anti-FLAG, anti-HSP90.5 and anti-HSP90.2 antibodies.