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. Author manuscript; available in PMC: 2017 Dec 5.
Published in final edited form as: Dev Cell. 2016 Nov 23;39(5):597–610. doi: 10.1016/j.devcel.2016.10.020

Figure 4. EBF1 and EBF2 directly associate with both the Pr and Pfr forms of phyB.

Figure 4

(A) LCI assay of the interactions of EBF1 and EBF2 with phyB in tobacco leaves. Full-length of EBF1 and EBF2 were fused to the split C-terminal (LUCc) fragments of luciferase. The full-length sequence, N-terminal and C-terminal fragments of phyB (phyB, phyBN, phyBC) were fused to the split luciferase N-terminus (LUCn). Empty vectors were used as negative controls. C.P.S is counts per second. Mean ± s.d., n=5.

(B and C) Co-immunoprecipitation assays for the interactions of EBF1 (B) or EBF2 (C) with phyB. Four-day-old dark-grown WT and transgenic seedlings over-expressing EBF1-TAP or EBF2-TAP seedlings were maintained in the dark (D) or exposed to red light (D-R) for 30 min. Proteins were extracted and immunoprecipitated using IgG Sepharose and immunoblotted using the indicated antibodies. Actin was used as a loading control.

(D) Pull-down assays to examine direct protein interactions in vitro. Recombinant phyB incubated with phycocyanobilin (Pfr) or methanol alone (Pr) was used as prey and pulled down using purified MBP, EBF1-MBP or EBF2-MBP proteins as bait.