Figure 8.

BIN2 Interacts with PUB40 In Vitro and In Vivo.

(A and B) Both BL (A) and Bikinin (B) reduce PUB40 stability. 35S-PUB40-YFP was treated with Bikinin (30 μM) or BL (50 nM) for 3 h. PUB40-YFP and BZR1 levels were detected with anti-YFP and anti-BZR1 antibodies, respectively.

(C) PUB40 interacts with BIN2 in yeast cells. The BD fused to PUB40, the AD fused to BIN2, and AD constructs were cotransformed into yeast cells as indicated. Yeast cells were grown on SD or SD-His medium. BD, binding domain; AD, activation domain; SD, synthetic dropout.

(D) In vitro pull-down assay to test the interaction between PUB40 and BIN2. MBP-YFP or MBP-PUB40 was incubated with GST-BIN2–bound beads. Proteins pulled down by GST-BIN2 were analyzed using anti-MBP and anti-GST antibodies.

(E) BiFC assays to test the in vivo interaction between BIN2 and PUB40. The indicated constructs were cotransformed into wild-tobacco–leaf epidermal cells. The BZR1-nYFP and BIN2-cYFP pair was used as a positive control in the BiFC assay.

(F) Coimmunoprecipitation between PUB40 and BIN2 in vivo. BIN2-YFP protein was immunoprecipitated by anti-YFP antibody from wild tobacco cells coexpressing PUB40-myc and BIN2-YFP and detected with anti-YFP and anti-myc antibodies.