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. 2011 Jan 31;10(4):M110.002642. doi: 10.1074/mcp.M110.002642

Fig. 7.

Fig. 7.

14-3-3 binding to CBX4 plays a role in the interaction between CBX4 and BMI1/PCGF family members but has no role on CBX4 localization at Polycomb bodies. A, Sequence alignment shows that a putative 14-3-3-binding site is conserved among the CBX4 protein sequences from various vertebrates. B, Mutation of the putative 14-3-3-binding site in CBX4 (CBX4[S164A]) abolishes 14-3-3-CBX4 interaction in a co-immunoprecipitation assay. HEK293 were transfected to express transiently HA-tagged CBX4 and CBX4[S164A] mutant proteins together with GFP-tagged 14-3-3ζ and 14-3-3ε. Proteins were immunoprecipitated (IP) with an anti-HA antibody linked to agarose beads, separated by SDS-PAGE, and detected (WB) using the indicated αGFP or αHA antibodies. Levels of protein expression were analyzed in total lysates. C, Mutation of the putative 14-3-3-binding site in CBX4 reduces CBX4 interaction with PCGF6 and BMI1, but not with RNF2 in a co-immunoprecipitation assay. HEK293 were transfected to transiently express HA-tagged CBX4 and CBX4[S164A] proteins together with GFP-tagged RNF2, PCGF6, and BMI1. Proteins were immunoprecipitated (IP) with an anti-HA antibody linked to agarose beads, separated by SDS-PAGE, and detected (WB) using the indicated αGFP or αHA antibody. Levels of protein expression were analyzed in total lysates. D, Mutation of the putative 14-3-3-binding site in CBX4 reduces CBX4 interaction with BMI1, whereas deletion of the Pc box abolishes the CBX4-RNF2 interaction in a co-immunoprecipitation assay. HEK293 were transfected to transiently express HA-tagged wild type CBX4, mutant CBX4[S164A] and deletion mutant CBX4[1–269] proteins together with GFP-tagged RNF2 and BMI1. Proteins were immunoprecipitated (IP) with an anti-HA antibody linked to agarose beads, separated by SDS-PAGE, and detected (WB) using the indicated αGFP or αHA antibodies. Levels of protein expression were analyzed in total lysates. E, Schematic representation of the CBX4 constructs used. The chromodomain is drawn as a blue triangle, the Pc box as a red circle, and the S164A mutation as a cross. F, CBX4 Pc box, but not the chromodomain, is required for CBX4 localization at Polycomb bodies. U2-OS cell lines stably expressing GFP-tagged CBX4 as well as mutated versions CBX4[S164A], CBX4[1–269] and CBX4[68–290] were fixed with paraformaldehyde and localization of fusion proteins visualized by fluorescence microscopy (upper panels). Hoechst staining labels nuclei (lower panels).