Fig. 6. Proteasome activity is required for the ectopic induction of AP3 expression in seedlings expressing 35S::UFO and 35S::LFY-GR. (A) The hlr mutation abrogates DEX-dependent induction of AP3 expression. Levels of AP3 expression monitored by qRT-PCR and normalized to EF-1. (B) Epoxomicin treatment reduces DEX induced AP3 expression in seedlings that express 35S::UFO-Myc and 35S::LFY-GR. Gray and white bars represent two different biological replicates. Levels of AP3 expression monitored by qRT-PCR and normalized to GAPDH. Ten plants assayed for each condition. Values represent mean±s.e.m. for the three technical replicates. (C) A subpopulation of LFY-FLAG protein is post-translationally modified and stabilized in the presence of epoxomicin. SDS-PAGE of LFY-FLAG protein as detected by FLAG antibodies shows a prominent band of LFY-FLAG protein at approximately 55 kDa (lane 1), as well as a smear of higher molecular weight proteins. The levels of these higher molecular weight proteins are reduced in a ufo-2 mutant background (lane 2). Conversely, an increase in the levels of these higher molecular weight proteins is seen in epoxomicin-treated 35S::LFY-FLAG inflorescences (lane 4) when compared with mock (DMSO)-treated tissue (lane 3). No crossreacting proteins are detected in wild-type samples (lane 5). Blot was reprobed with RPN6 as a loading control. (D) LFY-FLAG protein is ubiquitylated. (Top panel) Immunoprecipitated LFY-FLAG protein or control IgG probed with anti-ubiquitin antibodies; (bottom panel) probed with anti-FLAG antibodies. Protein extracts from wild-type (WT), 35S::LFY-FLAG (LF) or 35S::LFY-FLAG; ufo-2 (LF; ufo2) inflorescences. LFY-FLAG protein migrates at 55 kDa (arrowhead), while polyubiquitylated species can be detected in the 150-220 kDa range (bracket). A 115 kDa UFO-dependent band can be detected (asterisk) in the LF lane, suggesting LFY-FLAG is post-translationally modified in a UFO-dependent manner.