Fig. 4: LRBs interact with CRY2 in vivo.
From: Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases
a Confocal microscopic images showing BiFC signals of indicated protein pairs transiently expressed in Arabidopsis leaves. The Hoechst 33342 (which is used to stain the nuclei) and GFP (BiFC) signals are shown. The relative intensity of CRY2/LRB interaction was presented as BiFC ratio, calculated as BiFC ratio = [GFP intensity]nuclei / [Hoechst 33342 intensity]nuclei. BiFC ratio (mean ± SD), total number of quantified images and nuclei are shown blow the images. CRY2D387A, photo-insensitive CRY2, is used as the negative control. Scale bars, 10 μm. b Split-luciferase complementation assays showing the interactions of LRB1 and CRY2 or LRB2 and CRY2 in tobacco. Indicated split-LUC protein pairs were transiently co-expressed in N. benthamiana with FGFP-PPK1. LRB1ΔBTB, LRB1 with deletion of BTB domain (143-212 aa); LRB2ΔBTB, LRB2 with deletion of BTB domain (145-250 aa). c Quantification of split-luciferase complementation assays shown in (b). Data was shown as mean ± SD (n = 3 individual experiments). d–e Co-immunoprecipitation assays showing the interactions of LRB1 and CRY2 (d) or LRB2 and CRY2 (e) in Arabidopsis. 7-day-old seedlings expressing Myc-LRB or co-expressing FGFP-CRY2 and Myc-LRB were grown in the dark and then treated with blue light (100 μmol m−2 s−1) for the indicated time, or grown in long days (LD, 16 h light/ 8 h dark) and continuous white light (cWL). Plant extracts were immunoprecipitated by GFP-trap beads. The IP and co-IP products were detected with anti-Flag and anti-Myc antibodies, respectively. Co-IP experiments of 7-day-old seedlings co-expressing FGFP-CRY2 and Myc-CRY2 were performed in parallel as positive controls. The relative interaction intensity of CRY2/LRB or CRY2/CRY2 is calculated by [Co-IP intensity] / [IP intensity] and shown blow the immunoblots. The above experiments were repeated twice with similar results.
