Figure 6. KIB1-BIN2 interaction blocks BIN2 accessing its substrate BZR1.

(A) KIB1C-YFP transgenic Arabidopsis grown under the light for 5 weeks shows stem bending.

(B) KIB1C-YFP transgenic Arabidopsis shows constitutive BR-response phenotypes. Left panel: seven-day-old seedlings of WT and KIB1C-YFP grown in the dark on the medium with or without 2 µM PPZ. Scale bar is 10 mm; right panel: the quantitation of hypocotyl lengths in the indicated genotypes. The data are shown as means ± SD, n=15.

(C) Immunoblot analyses of BZR1, BIN2 and phospho-GSK3 using the anti-BZR1, anti-BIN2, anti-pTyr279/Tyr216 (pTyr) or anti-Tubulin antibody from ten-day-old seedlings of WT and KIB1C-YFP grown on the medium containing 2 µM PPZ (−) or 100 nM BL (+). Nonspecific bands (NS) and Tubulin protein level show the equal loadings. The numbers inside the images indicate the relative ratios of the signal intensity between BIN2 or pTyr and tubulin bands. The ratio of WT was set to 1.

(D) In vitro pull down assay shows KIB1 blocks BIN2 binding to BZR1. The mixtures of the indicated proteins BZR1-HA, KIB1-HA or YFP-HA were pulled down by BIN2-GFP-HA immobilized on the GFP-nAb magnetic agarose beads, and analyzed by immunoblots using an anti-HA antibody. YFP-HA was used as a negative control.

(E) Co-immunoprecipitation assay shows BR enhances BIN2-KIB1C interaction. Protein extracts from seven-day-old light-grown KIBC-YFP seedlings grown on the medium containing 2 µM PPZ treated with 100 nM BL or mock solution for 10 minutes were immunoprecipitated using an anti-BIN2 antibody, and analyzed by immunoblots using an anti-GFP antibody.

(F) Inhibiting kinase activity of BIN2 by bikinin causes BIN2 degradation. Wild-type seedlings were grown on the medium containing 2 µM PPZ under the light for 10 days, and then treated with 100 nM BL or 30 µM bikinin for the indicated time. Proteins were immunoblotted using an anti-BIN2 antibody. Ponceau S staining bands show the protein loadings.

Also see Figure S7.