Figure 1: ABI1 degradation is mediated by the 26S proteasome pathway.
From: Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases
(a) Treatment with the 26S proteasome inhibitor MG132 greatly increases the level of ABI1. Wild-type seedlings were treated with 50 μM MG132 or H2O for 6 h, or 50 μM ABA plus 50 μM MG132 or H2O for 6 h, and then total proteins were extracted and used for immunoblotting analysis with anti-ABI1 antibody. ACTIN protein was used as a loading control. (b) Quantitative analysis of the band intensity in a. The abundance of ABI1 at the start (ABA-, MG132-) was set to 1 as a reference for calculating relative abundance of various treatment. Error bars means±s.e.m. (n=3 independent experiments). (c) ABI1 degradation is enhanced by addition of ATP. Wild-type seedlings were treated with 50 μM ABA for 6 h, then total proteins were isolated and incubated with or without 1 mM ATP for different times, and subjected to immunoblotting analysis with anti-ABI1 antibody. ACTIN protein was used as a loading control. (d) Quantitative analysis of the band intensity in c. The abundance of ABI1 at the 0 min (ATP−, ATP+) was set to 1, respectively. The values were references for calculating relative abundance after various treatment time. Error bars are means±s.e.m. (n=3 independent experiments). (e) Addition of the protein biosynthesis inhibitor cycloheximide (CHX) does not change the degradation pattern of ABI1. Wild-type seedlings were treated with 50 μM ABA for 6 h firstly. After washing away excess of ABA, the seedlings were treated with 100 μM CHX or 50 μM MG132 separately for different times before protein was isolated for western blot with anti-ABI1 antibody. ACTIN was used as a loading control. (f) Quantitative analysis of the band intensity in e. The abundance of ABI1 at the 0 h (CHX, MG132) was set to 1, respectively. The values were references for calculating relative abundance after various treatment time. Error bars are means±s.e.m. (n=3 independent experiments).
