Figure 3: The two HAB1 isoforms have different levels of phosphatase activity and different effects on OST1 activity.
From: ABA signalling is fine-tuned by antagonistic HAB1 variants
(a) Subcellular localization analysis of HAB1.1 and HAB1.2 in the transient transformation of N. benthamiana leaf cells. Scale bar, 20 μm. (b) Comparison of the phosphatase activity of the two HAB1 isoforms in vivo. Recombinant HAB1.1 and HAB1.2 proteins were purified in E. coli and 100 ng proteins were used for the phosphatase activity assay. The s.d. of three experiments is shown using error bars. (c) Interaction between the HAB1 isoforms and OST1 in a BiFC assay. The HAB1.1-YFPC and HAB1.2-YFPC were co-transformed into N. benthamiana leaf cells with OST1-YFPN. At 48 h after transformation, the transformed leaves were stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min and used for the fluorescence observation. (d) Pull-down assay of the HAB1.2 and OST1 interaction. Equal amounts of the purified OST1–MBP fusion protein was added to tubes containing c-Myc-Agarose beads and c-Myc-Agarose beads plus HAB1.2-c-Myc protein, respectively. Anti-MBP antibody was used to detect the HAB1.2–OST1 interaction. (e) Effects of HAB1.1 and HAB1.2 on OST1 phosphorylation activity. OST1 protein was mixed with different concentrations of HAB1 isoforms for 30 min in kinase buffer and the auto-phosphorylation activity of OST1 was detected using phosphorimager. (f) Structures of the HAB1 isoforms predicted by the ESyPred3D software.
