Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - APB motif binds specifically to phyB (Pfr) under continuous Rc treatment, compensating for any potential dark reversion of the phytochromes during the coincubation. Coimmunoprecipitation of all five, recombinant phys (as prey) was tested using either GAD (control) or GAD:APB (PIF3) fusion proteins as bait, under continuous Rc during the coimmunoprecipitation assays. (A) Prey input (phyA, phyB, phyC, phyD, and phyE) proteins used for in vitro coimmunoprecipitation assays. (B) through (D) show the pellets from the coimmunoprecipitations. (B) As a control for the shorter coincubation period used here than for our routine assay conditions, the interaction of phyB (Pfr) with GAD or GAD:APB (PIF3) was tested after 20 minutes incubation in the dark at 4^oC following a red light pulse. (C) and (D) Photoactivated phyA, phyB, phyC, phyD, or phyE were used as prey with 20 minute incubation under continuous Rc (58 μmoles m^-2s^-1) (C) or Rc (116 μmoles m^-2s^-1) (D). (E) Quantitative comparison of in vitro coimmunoprecipitation assays using GAD:APB and phyB (Pfr) incubated for 20 minutes either in dark or Rc as indicated.
• Supplemental Figure 2 - Quantitative binding curves indicating lower apparent affinity of the PIL1-APB motif for phyB than the PIF3-APB motif. (A) Increasing amounts of phyB (Pfr) prey input (as indicated) were used in in vitro coimmunoprecipitation assays using GAD (control), GAD:APB(PIF3), or GAD:APB(PIL1) fusion proteins as bait. Phosphoimager images of the pellets are shown. (B) Quantification of in vitro coimmunoprecipitation assays. Bound phyB (fmoles)/fmole of bait was calculated and plotted against the increasing amounts of phyB input (fmoles).