Figure 3.

CRY1 interacts with RGA and GAI in a blue light-dependent manner. A, B, Pull-down assays showing the interactions of CRY1, CNT1, and CCT1 with RGA (A) and GAI (B). His-TF-CRY1, His-TF-CNT1, and His-TF-CCT1 served as baits and were detected with anti-His antibody. GST–RGA and GST–GAI served as preys and were detected with anti-GST antibody. Two independent experiments were performed, and one is shown. C, D, Split-luciferase complementation imaging assays showing the interaction of CRY1 with RGA (C) and GAI (D). Venus-nLUC and cLUC-Venus served as negative controls. Two independent experiments were performed, and one is shown. E, F, Cell-free GST pull-down assays showing the blue light-specific interaction of CRY1 with RGA (E) and GAI (F). GST–RGA and GST–GAI served as baits. Preys were protein extracts prepared from Myc-CRY1-OX seedlings that were dark-adapted and exposed to blue light (BL, 30 μmol·m^–2·s^–1), red light (RL, 50 μmol·m^–2·s^–1), or far-red light (FR, 10 μmol·m^–2·s^–1) for 1 h. Two independent experiments were performed, and one is shown. G–J, co-IP assays showing the blue light-dependent interaction of CRY1 with RGA and GAI. Dark-adapted seedlings of RGA-Flag-OX in WT (G) or cry1 (H), and GAI-Flag-OX in WT (I) or cry1 (J) were maintained in the dark or exposed to blue light (50 μmol/m²/s) for 1 h, followed by immunoprecipitation with an anti-CCT1 antibody. The IP (CRY1) and co-IP signals (RGA and GAI) were detected in immunoblots probed with anti-CCT1 and anti-Flag antibodies, respectively. Two independent experiments were performed, and one is shown.