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. 2013 Nov 1;27(21):2367–2379. doi: 10.1101/gad.226621.113

Figure 4.

Figure 4.

TAP of Sfmbt protein complexes from Drosophila embryonic nuclear extracts identifies a larger PhoRC assembly that resembles mammalian PRC1.6/E2F.6 complexes. (A) Sfmbt complexes isolated by TAP from wild-type (wt) or α-tubulin-Sfmbt-CTAP transgenic embryos. (Left) Input material for purification was normalized by protein concentration, and equivalent amounts of eluate from calmodulin affinity resin were separated on a 4%–12% polyacrylamide gel and visualized by silver staining; the molecular weight marker is indicated on the left. Sfmbt bait protein containing the calmodulin-binding tag (Sfmbt-CBP), its degradation products, and bands representing Mga (CG3363), Hdac1/Rpd3, Pho, Nap1, and HP1b were identified by MS (Supplemental Table S2). (Right) The Nap1 and HP1b proteins were undetectable as bands on silver-stained gels, and their presence was verified by Western blot analysis: total embryonic nuclear extract input material from wild-type (wt) and Sfmbt-CTAP transgenic embryos (lanes 1,2) and material eluted from the calmodulin affinity resin after purification (lanes 3,4), probed with the indicated antibodies. The Nap1 and HP1b panels come from the same batches of input and eluate material, and the same ratio of input versus eluate was loaded in both cases. (B) The Drosophila Sfmbt assembly resembles human PRC1.6/E2F.6 assemblies. Graphic representation of the larger Drosophila Sfmbt–Pho assembly with the additional proteins identified in A and therefore called PhoRC-L and the PRC1.6 assembly described in Gao et al. (2012). Note that the PRC1.6 assembly is identical to the E2F.6 assembly described in Ogawa et al. (2002) but was reported to also contain HDAC1/2 and WDR5. Drosophila Sfmbt and human L3MBTL2 proteins are labeled in green, the orthologous subunits identified in both Drosophila and human assemblies are labeled in blue, and Pho is labeled in orange. The Drosophila genome does not encode orthologs of E2F.6 and MBLR (asterisks), and this might explain why Drosophila PhoRC-L assemblies do not contain the RING1A/B ortholog Sce, the RYBP/YAF2 ortholog Rybp, and the DP-1 ortholog Dp.