Figure 4.

Light Induces Rapid Phosphorylation and Ubiquitination prior to Degradation of PIF1.

(A) LUC-PIF1 shows high molecular weight bands (LUC-PIF1-ubi) after Rp (3000 μmol·m⁻²). A blot probed with anti-LUC antibody is shown.

(B) TAP-PIF1 shows high molecular weight bands (TAP-PIF1-ubi) and is also degraded following Rp (100 μmol·m⁻²), while TAP-GFP is stable under these conditions. A blot probed with anti-MYC antibody that recognizes the TAP tag is shown. Asterisks denote cross-reacting bands.

(C) and (D) The Rp- and FRp-induced slow-migrating bands are ubiquitinated forms of PIF1. TAP-PIF1 was immunoprecipitated from protein extracts prepared using 4-d-old dark-grown seedlings either kept in the dark or exposed briefly to Rp light (3000 μmol·m⁻²; [C]) or FRp light (3000 μmol·m⁻²; [D]). The immunoprecipitated samples were then separated on 6.5% SDS-PAGE gels and probed with anti-Ubi or anti-MYC antibody.