Gfp agarose beads

For SIZ1-HLS1 interaction, 3-d-old dark-grown HLS1pro:HA-FLAG-HLS1 SIZ1pro:SIZ1-GFP and HLS1pro:HA-FLAG-HLS1 SIZ1pro:GFP transgenic seedlings were used for Co-IP assays (Zhang et al. 2021) . The total proteins were extracted from different seedlings and then incubated with GFP agarose beads (Chromotek) in IP buffer [10 mM Tris-HCl, pH 7.5, 0.5% (v/v) Nonidet P-40, 2 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1% (w/v) protease inhibitor]. The beads were collected and washed at least 5 times with IP buffer, then the interaction by immunoblotting was examined using anti-HA (1:5,000 dilution, Sigma-Aldrich, USA, H6908) and anti-GFP (1:5,000 dilution, Transgen, China, HT801) antibodies. For HLS1-phyB interaction, HA-HLS1, MYC-SUMO1, GFP, and phyB-GFP plasmids were transformed into Arabidopsis protoplasts isolated from Col-0 or siz1-2. Arabidopsis protoplasts were prepared as described previously (Yoo et al. 2007 (link)). After 16 h of expression in the darkness, protoplasts were transferred to light for 2 h, and then proteins were extracted and incubated with HA agarose beads in IP buffer. The anti-HA, anti-MYC (1:5,000 dilution, Cwbio, China, cw0299M), and anti-GFP antibodies were used for immunoblotting. The antibody source details are listed in Supplemental Table 1.

The ChIP assay was performed as previously described (Shi et al., 2012) with minor modifications. WT LH244 and bZIP68-GFP overexpressing seedlings were grown at 25 C in the greenhouse for 14 days under a 16-h light/8-h dark photoperiod and treated with or without cold at 4 C for 12 h. The leaves were crosslinked and chromatin was extracted from the cross-linked samples and fragments with an ultrasonicator. DNA fragments associated with bZIP68-GFP protein were incubated with GFP agarose beads (ChromoTek). The enriched DNA fragments were collected with a ChIP DNA Clean and Concentrator kit (ZYMO). Immunoprecipitated DNA was quantified by qPCR using the primers of target genes. Relative enrichment was represented by input (%). The primers are listed in Supplemental Data Set S10.

Co-IP assays were performed as described previously (Ding et al., 2015) . The plasmids Super:MPK8-MYC and Super:bZIP68-GFP were transformed into Agrobacterium GV3101 and co-infiltrated into N. benthamiana leaves, followed by incubation at 25 C for 36 h. The empty-vector Super:GFP was used as a negative control. Total proteins were extracted from the samples with protein extraction buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 20% glycerin, 0.1% NP-40, 1 mM DTT, and 1 protease inhibitor cocktail) and incubated with GFP agarose beads (ChromoTek) for 2.5 h. The immunoprecipitated samples were washed five times with washing buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 20% glycerin, 0.1% NP-40, 0.1% Triton X-100) and subjected to immunoblot analysis. The protein interaction signal was detected with anti-MYC antibody (Sigma-Aldrich).