Genes involved in ubiquitin-proteasome pathway are identified as direct RIN targets. (a) Expression analysis of SlUBC32 and PSMD2 at protein and mRNA levels. The protein expression in wild-type (WT) and rin mutant was assessed by quantitative proteome analysis at breaker (Br) and orange (Or) ripening stages. The mRNA expression was examined by quantitative RT-PCR. The gene transcript levels are normalized against the actin gene. Values are means ± SD of three independent experiments. (b) ChIP-qPCR assays show that RIN direct binds to the promoter regions of SlUBC32 and PSMD2. The promoter structures of the target genes are presented. Blue boxes represent CArG box elements and numbers indicate the position of these motifs relative to the translational start site. Green fragments with upper-case letters represent the regions used for ChIP-qPCR. Values are the percentage of DNA fragments that co-immunoprecipitated with anti-RIN antibodies (black bars) or non-specific antibodies (preimmune rabbit IgG; grey bars) relative to the input DNAs. Error bars represent the SD of three independent experiments. (c) Gel mobility shift assays reveal the direct binding of RIN to CArG box elements in the promoter regions of SlUBC32 and PSMD2. The probe sequences corresponding to the SlUBC32 and PSMD2 promoters are shown, with red letters representing the CArG box. The mutated bases in the probes are represented by blue letters. wt, probe with intact CArG box element; mt, probe with mutated CArG box element. As competitors, 1,000-fold excess amounts of unlabeled probes were added to the binding reaction. The retarded bands and the free probes are indicated by arrowheads.