Fig. 2: Spatio-temporal expression patterns of CDF2 and PIF4, and physical interaction between the two proteins.
a,b, RT–qPCR analysis of levels of HA-CDF2 (a) and PIF4-HA (b) mRNA. Data are represented as means ± SEM of three independent amplifications. All values are normalized to APA1 levels. c,d, Western blots comparing the accumulation of HA-CDF2 (c) and PIF4-HA (d) proteins. Time (h) is expressed as hours from dawn (ZT, zeitgeber). Actin served as the loading control. RNA and protein were extracted throughout a SD in 6-day-old seedlings. Western blots in b and c were performed twice with similar results. e,f, Confocal microscopy analysis of epidermal cells of 6-day-old CDF2::CDF2-mVenus (e) and PIF4::mScarlet-I-PIF4 (f) transgenic plants grown under SD. Scale bars, 30 μm. g, HA-CDF2 protein co-immunoprecipitates with PIF4-TAP (9×Myc-6×His-3×Flag) from 6-day-old SD-grown seedlings. Co-IP experiments in b and c were performed twice with similar results. h, Box diagram of various fragments of CDF2 and PIF4 used in i. i, PIF4-Myc (C-terminal) interacts with HA-CDF2 (N-terminal) in vitro in the light. PIF4-Myc, HA-CDF2 and their truncation proteins were synthesized in a cell-free system. In vitro pull-down assays in i were performed three times with similar results.
