Anti abi5

Seeds of wild-type Col-0 and OX70-3, which were grown in the same growth chamber and harvested at the same day, were sown on MS medium containing 5 μM ABA and cultivated in a growth chamber under continuous white light for 3 d. After being washed twice with liquid MS medium, the seeds were transferred to fresh liquid MS medium supplemented with or without 50 μM MG132 (Sigma-Aldrich) or 100 μM CHX (Sigma-Aldrich), and harvested at the indicated time points. Subsequently, total proteins were extracted from the samples using SDS lysis buffer (Beyotime) and accurately quantified. After mixing with 5 × SDS-loading buffer, the proteins were boiled for 10 min, isolated on a 10% SDS-PAGE gel, and subsequently transferred to polyvinylidene fluoride (PVDF) membrane and incubated in blocking buffer (20mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk powder) for 1 h at room temperature, then with 1:2000 anti-ABI5 (Abcam) or 1:1,000 anti-RPN6 (Enzo) for 12 h at 4°C, and followed with HRP-conjugated goat anti-rabbit IgG (Thermo) for 1 h. Finally, the PVDF membrane was overlaid on high-sig ECL western blotting substrate (Tanon) for 5 min and imaged using a CHEMI DOC^TMXRS (Bio-rad).

Anti-Myc, anti-HIS, anti-GFP, and anti-RFP antibodies were purchased from Easy Bio System Inc.; anti-LUC was purchased from Sigma-Aldrich; anti-HA was purchased from CoWin Biosciences; anti-ABI5 was purchased from Abcam; anti-Flag, anti-GST, and anti-MBP antibodies were purchased from ProteinTech; anti-UBP12 was gifted by X. Cui from the Chinese Agricultural Academy of Sciences; and anti-Ub and anti-PYL4 antibodies were generated and purified from the stock of our laboratory.