Figure 4. The nuclear-resident phyA-signaling intermediates FHY3 and FHY1 preferentially associate with underphosphorylated phyA in vivo.

(A) Immunoblot analysis of purified nuclear (N) and nuclei-depleted soluble (S) fractions from 5-day-old wild type seedlings grown under the indicated light conditions: D, continuous darkness; FR, FRc; D–R, D followed by Rc for 10 min. An approximately 12-fold quantity (in protein content) of the nuclear fraction compared to the nuclear-depleted fraction on a per-tissue amount basis was loaded. Fraction markers used were histone H3 (Histone), cytosolic HSP90 and phosphoenolpyruvate carboxylase (PEPC). Specificity of anti-FHY3 antibodies was verified (right). (B) Co-IP of phyA with the indicated antibodies using FRc-grown seedlings. Respective pre-immune serum was used as an IP control (Pre). The Rpt5 immunoblots are shown to verify co-IP specificity. (C) R-induced phyA-FHY3 dissociation. Design of experiments (top left): Total protein lysates of the FRc-grown phyA-overexpressing seedlings were irradiated with 5 min of FR light pulse (FR) or 5 min of FR light pulse immediately followed by 5 min of R light pulse (FR/R), and then subjected to co-IP analysis with anti-FHY3 and anti-COP1 antibodies (bottom). Immunoblot analysis of lysates of FRc-grown seedlings kept under Rc for 3 h ensured phyA stability during the procedure (top right). (D) COP1 co-IP analysis of FRc-grown seedlings including wild types (Col-0 and No-0), fhy3-1 far1–2, and fhy1-1. Relative band intensities normalized for each panel and the ratio of cap-phyA to underphosphorylated phyA are shown. The asterisk and arrowhead indicate cap-phyA and underphosphorylated phyA, respectively.