Cycloheximide c1988

Wild-type and mutant seedlings were germinated on 0.5X MS media (Murashige and Skoog, 1962) for 4 days. On day 5, 30 seedlings of each genotype were transferred to 24 well-plates containing 1 ml 0.5X MS supplemented with 50 M MG132 (474787, Sigma-Aldrich). Plates were sealed with surgical tape and placed on a shaker at 55 rpm for 16 h at 23 o C under continuous light. After 16 h, the seedlings were washed 3X with 1 ml fresh media. Cycloheximide (C1988, Sigma-Aldrich) (400 M in DMSO) or DMSO control was added at 0 h, and harvesting occurred at 0, 2, 4, 5, 8, 10 or 24 h, respectively. Samples of 30 seedlings were flash frozen in liquid nitrogen and stored at -80°C prior to protein extraction. Tissue was homogenized in liquid nitrogen, and hot SDS buffer (8 M urea, 2% SDS, 0.1 M DTT, 20% glycerol, 0.1 M Tris-Cl pH 6.8, 0.004% bromophenol blue) was added prior to SDS-PAGE and Western blotting. Anti-GFP (1:2000; 11814460001, Roche) served as the primary Ab, followed by secondary Goat Anti-Mouse IgG [HRP] (1:3000; A00160, Genscript). Proteins were detected with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) using an Azure 300 chemiluminescence imager (Azure Biosystems), and blots were stained with Bio-Safe Coomassie Blue G-250 (Bio-Rad) for loading controls. Band intensities were quantified ImageJ software.