Anti gfp agarose conjugate

Immunoblot and immunoprecipitation assays were performed as previously described with slight modifications (54 (link)). Briefly, 6-day-old seedlings were treated with 100 μM JA for 6 h, and then roots were collected and ground in liquid nitrogen. The CK2mut seedlings were pre-treated with 1 μM Dex for 24 h before JA treatment. Proteins were extracted in RIPA buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin]. Anti-GFP antibody (Santa Cruz) was used to detect MYC2-GFP proteins. For immunoprecipitation assays, protein extracts were pre-incubated for 1 h at 4°C with Protein A agarose (Roche), and then incubated for 4 h at 4°C with Anti-GFP agarose conjugate (Santa Cruz). Immunocomplexes were washed five times with RIPA buffer and eluted by adding 4 × SDS loading buffer followed by 5 min of boiling. The anti-phospho-(Ser/Thr) antibody (Abcam) was used to detect the phosphorylated isoform of MYC2. All the experiments were performed three times and showed similar results.

ChIP was performed as previously described (55 (link)). Anti-GFP agarose conjugate (Santa Cruz) was used to immunoprecipitate the protein-DNA complex. The isolated chromatin sample before precipitation was used as the input control. The enrichment of DNA fragments was determined by qRT-PCR. The relative enrichment was calculated by normalizing the amount of a target DNA fragment after ChIP against that of a genomic fragment of ACT2 (AT3G18780) before ChIP (Input). The binding of MYC2 with the G-box motifs in the promoter region of JAZ1 (–283 bp from ATG), JAZ3 (–343 and –401 bp from ATG), LOX3 (–254 bp from ATG), and CORI3 (–147 bp from ATG) was determined with specific primers listed in Supplementary Table S1. The experiments were performed in biological and technical triplicates.