Figure 7.
Post-translational decay of HFR1. (A) HFR1 decay is delayed by induced LAF1 expression. (Panel a) Four-day-old seedlings (35S-HFR1-3HA and XVE-LAF1-6Myc) grown in darkness were incubated in liquid MS medium with MG132 (50 μM) or MG132 (50 μM) plus β-estradiol (10 μM) for 12 h with continuous light, washed, and then transferred to MS medium with 100 μM cycloheximide (CHX). Treated seedlings were then incubated under FR light (1.5 μmol m−2 sec−1). Proteins were extracted at the indicated times and analyzed by Western blotting using anti-HA or anti-Myc antibodies. Tubulin levels were used as loading controls. Expression levels of HFR1 and tubulin were measured using the program Image Gauge version 3.12 (Fuji), and the values were normalized to 0 time in the “−” inducer or “+” inducer sample of both panels. (Panel b) Post-translational decay of LAF1. Four-day-old seedlings (35S-LAF1-3HA) grown in darkness were treated with MG132 (50 μM). All conditions and loading control were identical to those of panel a. (B) HFR1 decay is not delayed by induced LAF1(R97A) expression. (Panel a) Four-day-old seedlings [35S-HFR1-3HA and XVE-LAF1(R97A)-6Myc] grown in darkness were used, and all conditions were identical to those of A. (Panel b) Post-translational decay of LAF1 (R97A). Four-day-old seedlings [35S-LAF1(R97A)-3HA] grown in darkness were treated with MG132 (50 μM). All conditions and loading control were identical to those of panel a in A. (C) Post-translational decay of endogenous HFR1 in LAF1RNAi/Col. Four-day-old seedlings (wild type and LAF1RNAi/Col) grown in darkness were incubated in liquid MS medium with MG132 (50 μM), and other conditions were identical to those of A, except anti-HFR1 antibody was used. (D) Post-translational decay of LAF1 in hfr1-201. 35S∷LAF1-3HA/Col- or 35S∷LAF1-3HA/hfr1-201-overexpressing seedlings were used. All conditions were identical to those of C except anti-HA antibody.