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Fig. 1 | Epigenetics & Chromatin

Fig. 1

From: SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ

Fig. 1

Oligonucleotide-assisted complex purification of RBPJ and validation of a direct RBPJ-L3MBTL2 interaction. a Oligonucleotides used to stabilize RBPJ complexes during purification. The sequence of the double-stranded oligo is based on the Hes1 promoter as described in [27]. b Immunofluorescence of Flag-HA-tagged RBPJ expressed in HeLa-S cells. c Experimental outline of oligonucleotide-assisted complex purification of Flag-HA-RBPJ from HeLa-S cells. d Silver staining of purified RBPJ complexes, obtained in the presence and absence of the oligonucleotides. e Example proteins, that are strongly reduced in the RBPJ complex purified in presence of the oligonucleotides. f Proteins associated with the RBPJ coactivator or corepressor complexes. Components of the PRC1.6 are putative novel RBPJ-associated proteins. The numbers in e and f indicate the total peptide numbers identified by mass-spectrometry (see also Additional file 1: Table S1). g Schematic representation of PRC1.6 subunits [16]. h GST-pulldown assays were performed with GST-L3MBTL2 or GST only and [35S] methionine-labelled RBPJ. Bound proteins were separated in SDS-PAGE and visualized by autoradiography. i Co-Immunoprecipitation experiments were performed using Flag-L3MBTL2 and GFP-RBPJ overexpressed in HEK293T cells. GFP-RBPJ and Flag-L3MBTL2 were expressed in 293T cells. Lysates were subjected for GFP immunoprecipitation, followed by Western blotting. Control cells were transfected with pcDNA GFP plasmid

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