Fig. 5.
PP2A-A subunits may directly bind to SPCH. (A) Yeast two-hybrid assay for BD-SPCH with PP2A-A (AD-A1 and AD-A2). The BD and AD empty vectors were used as negative control, and SCRM/ICE1 was used as positive control. Yeast growth controls are shown on the Left (-Leu-Trp). Interaction tests are shown on the Right (-Leu-Trp-His with 2.5 mM 3-AT). (B) Confocal images of BiFC assays to test interactions between SPCH and PP2A-A subunits. The expression of half YFPs (YFPN and YFPC) were used as negative controls. SCRM/ICE1 was used as positive control. Yellow shows complemented expression of YFP signal. (Scale bars, 50 μm.) (C and D) In vitro pull-down assays by using E. coli-made recombinant proteins. Amylose resins-bound MBP, MBP-SPCH, or MBP-ICR1 were incubated with (C) His-A1 or (D) His-A2 proteins. MBP and MBP-ICR1 proteins were used as negative controls. Immunoblots were visualized by anti-His antibody. Input His-tagged proteins and MBP-tagged proteins were visualized by anti-His and anti-MBP antibodies, respectively. Red asterisks indicate the positions of related proteins.