Figure 2.
SIZ1 E3 Ligase Interacts with SEU and Regulates Its SUMOylation.
(A) Yeast two-hybrid assay. GAD, GAL4 activation domain; GBD, GAL4 DNA-binding domain; SIZ1-Δ, a truncated version of SIZ1 containing amino acids 1–664. −WLHA, without Trp, Leu, His, and Ade; −WL, without Trp and Leu.
(B) Semi-in vitro pull-down assay in N. benthamiana leaves.
(C)In vitro pull-down assay.
(D) Co-IP assay. Plants were grown under light (80 μmol/m2/s) for 5 d. Proteins were precipitated with an anti-GFP antibody conjugated to agarose beads.
(E) Detection of SUMOylated proteins in vivo. Proteins were precipitated with an anti-GFP antibody conjugated to agarose beads.
(F) Immunoblot analysis of SEU. Blotting with an anti-actin antibody served as a loading control.
(G) qRT–PCR analysis of SEU expression. Relative gene expression was normalized to the level of IPP2. Data are means ± SD of three biological replicates. Different letters indicate significant differences as determined by one-way ANOVA (P < 0.01). In (E)–(G), seedlings were grown under red light (40 μmol/m2/s) for 5 d.