Figure 6.

ChIP analysis of interactions between VIP1-CYP707A1/3 promoters in vivo. GFPox (VIP1-GFP −) or VIP1-GFPox (VIP1-GFP +) plants were rehydrated in 20 mm Tris-HCl, pH 6.8, for 10 min and used for the experiment. After making cross-links between VIP1-GFP and DNA, chromatin was isolated, sonicated, and used as an input for the subsequent PCR. VIP1-GFP was immunoprecipitated from the sonicated sample using a rabbit anti-GFP antibody. A rabbit anti-hemagglutinin (HA) antibody was used as a negative control. DNA was eluted from immunocomplexes subjected to the PCR analysis of CYP707A1/3 promoter fragments (CYP707A1p and CYP707A3p). Immunocomplexes were then disrupted by incubating them at 100°C in SDS sample buffer, and VIP1-GFP was analyzed by immunoblotting using the anti-GFP antibody (IB: GFP). In the combination of the anti-GFP antibody and VIP1-GFP + (lane 4), two bands were detected above the band of monomeric VIP1-GFP, suggesting that some VIP1-GFP-containing complexes were fixed by the cross-linking reaction. IgG HC, IgG H chain.