BBX28 requires COP1 to promote hypocotyl growth under shade. A) The Venn diagram shows co-regulated genes between BBX28 and COP1 under shade. The P-value of the intersection is indicated. B) Hypocotyl length of Col-0, bbx28-5, cop1-4, bbx28-5 cop1-4, cop1-6, 35S:YFP-BBX28 and 35S:YFP-BBX28/cop1-6 seedlings grown in a short-day photoperiod (L10:D14) at 22 °C for 2 d and maintained in white light (WL) or transferred to simulated shade (SS) treatment for 4 d. The boxplots show the median as a solid line and the mean as a black point, the hinges representing the 25th and 75th percentiles, and the whiskers that extend up to a maximum of 1.5 times the interquartile range from the hinges. Individual observations are represented by colored points (n > 50). Different letters indicate significantly different means obtained by two-way-ANOVA followed by the post-hoc Tukey's HSD (P < 0.05). C) Nuclear fluorescence in hypocotyl cells of YFP-BBX28 in Col-0 and cop1-6 seedlings grown in white light (WL) or simulated shade (SS) for 6 h. D) Nuclear fluorescence in hypocotyl cells of YFP-BBX28 in Col-0 seedlings grown in white light (WL) or simulated shade (SS) with DMSO or MG132 (100 μM) for 6 h. C and D) On the left, representative confocal images showing hypocotyl epidermal cells. The YFP signal is shown in green, and magenta represents the chlorophyll. Scale bars: 40 μm. On the right, mean fluorescence intensity. The boxplots show the median as a solid line and the mean as a black point, the hinges representing the 25th and 75th percentiles, and the whiskers that extend up to a maximum of 1.5 times the interquartile range from the hinges. Individual observations are represented by colored points (n = 12). Different letters indicate significantly different means obtained by two-way-ANOVA followed by the post-hoc Tukey's HSD (P < 0.05). E) Immunoblot analysis of GFP-BBX28 in seedlings grown as in (D). The BBX28 protein and the non-specific band used as a loading control are indicated. The position of the marker with known molecular weight are indicated on the left. The GFP-BBX28 protein levels are shown as the intensity band relative to the loading control under WL and SS. Data represents the mean of three biological replicates and the standard deviation. Different letters indicate a significant difference between means by two-way ANOVA followed by the post-hoc Tukey's HSD (P < 0.05).