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. 2016 Mar 14;44(12):5597–5614. doi: 10.1093/nar/gkw156

Figure 4.

Figure 4.

Interaction of ESD7 with PRC2 components. (A) Diagrams showing ESD7 protein and the fragments used in these analyses, POLA5 and POLA3. The N-terminal catalytic domain is indicated by the black box and the C-terminal domain (white box) includes the POLA5 fragment carrying a MIR region and the POLA3 fragment carrying a zinc finger domain. (B) Yeast two-hybrid experiment showing interaction of ESD7 fragments with EMF2 and CLF. One out of three independent co-transformants is shown in the picture growing onto -LWH, -LWHA and -LWHA media containing different 3AT concentrations. (C) In vitro binding assay of a fragment of CLF containing the SET domain fused to GST with POLA3 and POLA5 ESD7 radiolabelled fragments. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. (D) In vitro binding assays of the fusion proteins GST-POLA5 and His6-EMF2-VEFS containing different fragments of the VEFS domain (4AD and 5AD). The EMF2-derived proteins were revealed by western blot with an αHis antibody. Protein molecular weights are indicated. The lower bands in the input lanes correspond to His6-EMF2 VEFS degradation products. Arrows indicate the expected size for the corresponding fusion proteins. (E) In vivo co-IP of POLA5 and CLF proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-CLF protein in clf-16 mutant background, POLA5-HA (L4) and from 35S::GFP-CLF 35S::POLA5-HA lines in clf-16 mutant background. An αHA antibody was used to pull-down the protein complexes and CLF protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins. (F) In vivo co-IP of POLA5 and EMF2 proteins. Nuclei proteins were extracted from plants overexpressing functional GFP-EMF2 protein in emf2-2 mutant background, POLA5-HA and from 35S::GFP-EMF2 35S::POLA5-HA lines in emf2-2 mutant background. An αHA antibody was used to pull-down the protein complexes and EMF2 protein was detected using an αGFP antibody. Protein molecular weights are indicated. Arrows indicate the expected size for the corresponding fusion proteins.