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. 2021 Sep 1;33(12):3675–3699. doi: 10.1093/plcell/koab221

Figure 2.

Figure 2

VDAL interacts with PUB25 and PUB26, two predicted plant U-box Ub E3 ligases. A, VDAL interaction with PUB25 and PUB26 in Y2H assay. The interaction of BD-PUB26 and AD-BIK1 is used as a positive control. BD-At4g20360 and BD-At3g44110 are used as negative controls. Yeast cells were grown on different media for 4 days. B and C, VDAL interaction with PUB25 (B) and PUB26 (C) in CoIP assay. VDAL-MYC and PUB25-GFP or PUB26-GFP plasmids were cotransfected into Arabidopsis protoplasts and incubated for 16 h. Total proteins were extracted and immunoprecipitated with anti-GFP beads, then immunoblotting analysis was carried out with anti-MYC and anti-GFP antibodies. D and E, VDAL interaction with PUB25 (D) and PUB26 (E) in the pull-down assay. Proteins purified from E. coli were immunoprecipitated with anti-GST beads and detected with anti-GST and anti-HIS antibodies. F, VDAL interaction with PUB25 and PUB26 in the split firefly LCI assay. Different plasmids combinations were transiently expressed in tobacco leaves for 72 h, then images were collected by a CCD camera.