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. 2021 Apr 19;33(7):2360–2374. doi: 10.1093/plcell/koab112

Figure 4.

Figure 4

NF-YCs interact with BIN2 in vitro and in vivo. A, Yeast two-hybrid assays showing the interaction between NF-YC9 and BIN2. Transformed yeast cells were grown on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu media. The interaction between BIN2 and BZR1 was used as a positive control. B, In vitro pull-down assay showing the direct interaction between His-NF-YC9 and GST-BIN2 recombinant proteins. His-tagged proteins were incubated with GST-BIN2 or GST proteins and the immunoprecipitated fractions were detected by anti-His and anti-GST antibodies. Arrow and arrowhead indicate GST-BIN2 and GST protein bands, respectively. C, BiFC analysis showing the interaction between NF-YC9 and BIN2 in Arabidopsis protoplasts. YFP, fluorescence of enhanced yellow fluorescent protein; mCherry, mCherry was fused with a nucleus localization signal (VirD2NLS-mCherry) as the nucleus indicator; Merge, merged of YFP and mCherry signals. The GUS gene served as a negative control. D, Co-IP assay of the interaction between NF-YC9 and BIN2 in planta. Nuclear extracts from 3-d-old nf-yc9-1 NF-YC9pro:NF-YC9-3FLAG/35Spro:BIN2-6MYC seedlings grown in the light were immunoprecipitated by anti-MYC antibody or preiummune serum Immunoglobulin G (IgG). The co-immunoprecipitated proteins were detected by anti-MYC or anti-FLAG antibody.