Fig. 2.
BBX32 physically interacts with COL3. (A) Y2H assay between BBX32 and each of COL3, COL3-N, and COL3-C. These experiments were repeated twice. (B) BiFC in tobacco. COL3-YFPC interacts with BBX32-YFPN in the nucleus and localized to nuclear speckles. 35S:GFP, p19 and empty vector (MCS-N MCS-C) were used as negative controls. B32-N MCS-C represents the fusion of BBX32 on N terminus alone; MCS-N COL3-C represents the fusion of COL3 on C terminus alone (Materials and Methods). Enlarged image of the nuclei shows the size and number of speckles. BF, bright field. (C and D) Co-Immunoprecipitation (Co-IP) in tobacco and Arabidopsis, respectively. Immunoprecipitations (IPs) were performed on tobacco leaves (3 dpi) and 10-d-old Arabidopsis seedlings grown in long days (16L: 8D) at 22 °C. Tissues were harvested 1 h after dawn. IP is performed by using anti-FLAG antibody and COL3 was coimmunoprecipitated with anti-GFP antibody. A 5% input was used. Western blots were performed on 10% (wt/vol) precast gels (Bio-Rad), and experiments were repeated at least five times.