Ously growing 293 cells were collected in lysis buffer. Immunopreciptations were performed with a polyconal antibody recognizing E2F6. Immunoprecipitated proteins were MNS biological activity resolved on SDS PAGE and assayed by Western blotting with a monoclonal antibody recognizing BRG1. D) HA-tagged E2F6 interacts with flag-tagged BRG1. Plasmid constructs overexpressing epitope-tagged versions of E2F6 and BRG1 were individually transfected or cotransfected into 293 cells. Immunoprecipitations were carried out with a polyclonal antibody recognizing the HA epitope tag on E2F6. Immunoprecipitates were resolved on SDS PAGE and Western blotted with the monoclonal M2 anti-flag antibody. doi:10.1371/journal.pone.0047967.glibrary using a full-length E2F6 clone as bait [18]. From this screen, we identified 14 independent clones that represented previously annotated proteins with a potential to regulate gene transcription (Table 1). Among these 14 clones, three clones containing fragments representing EPC1, DP1 and DP2 were identified [1,18,19,24]. Because these proteins have been shown to previously interact with E2Fs, this provided a strong validation 
of the screen. One additional clone contained a partial sequence coding for amino acids 462?78 of the BRG1 protein. Given that prior work has suggested a role for BRG1 in facilitating transcriptional regulation by a wide variety of proteins, we cloned full-length BRG1 and further confirmed its Licochalcone-A site interaction with E2F6.E2F6 immunoprecipitates with BRGTo determine an interaction between BRG1 and E2F6, we first incubated S35-labeled in vitro translated BRG1 with an E2F6glutathionine S transferase (GST) fusion protein. Precipitation with GST beads revealed in vitro translated S35-labeled BRG1 associated with GST-E2F6 but not GST alone (Figure 1a). To confirm an interaction between E2F6 and BRG1 in cells, wecoexpressed E2F6 and BRG1 in T98G cells. E2F6 was shown to immunoprecipitate with BRG1 when an antibody recognizing BRG1 was used (Figure 1b). Given that we saw an interaction between BRG1, we also tested the ability of endogenous E2F6 and BRG1 proteins to interact under normal physiological conditions in another cell line. In agreement with our studies above, an antibody recognizing endogenous E2F6 was able to immunoprecipitate BRG1 in 293 cells (Figure 1C). To confirm the interaction between BRG1 and E2F6 was not resulting from a cross reaction between the antibodies recognizing E2F6 and BRG1, we determined an interaction between epitope tagged E2F6 and BRG1 proteins using antibodies to HA and Flag. An antibody recognizing HA tagged-E2F6 was able to immunoprecipitate Flag tagged-BRG1 only in cells that coexpressed HAE2F6 and Flag-Brg1 and not in cells expressing either protein alone (Figure 24195657 1D). We quantitated the blots by densitometry to obtain an approximation of the fraction of proteins bound (Table S1).E2F6 and BRG1 in Transcriptional RegulationFigure 2. BRG1 interacts with the E2F4 and E2F6. BRG1 specifically interacts with E2F4 and E2F6 but not with the activator E2Fs. A plasmid construct expressing BRG1 was cotransfected into 293 cells with plasmid constructs expressing HA-tagged E2F1, E2F2, E2F3, E2F4 or E2F6. Immunoprecipitation experiments were carried out in lysis buffer with a polyclonal antibody recognizing the HA epitope tag. Immunoprecipitates were resolved on SDS PAGE and Western blotted with a BRG1 monoclonal antibody. doi:10.1371/journal.pone.0047967.gBRG1 specifically interacts with E2F4 and E2FNumerous.Ously growing 293 cells were collected in lysis buffer. Immunopreciptations were performed with a polyconal antibody recognizing E2F6. Immunoprecipitated proteins were resolved on SDS PAGE and assayed by Western blotting with a monoclonal antibody recognizing BRG1. D) HA-tagged E2F6 interacts with flag-tagged BRG1. Plasmid constructs overexpressing epitope-tagged versions of E2F6 and BRG1 were individually transfected or cotransfected into 293 cells. Immunoprecipitations were carried out with a polyclonal antibody recognizing the HA epitope tag on E2F6. Immunoprecipitates were resolved on SDS PAGE and Western blotted with the monoclonal M2 anti-flag antibody. doi:10.1371/journal.pone.0047967.glibrary using a full-length E2F6 clone as bait [18]. From this screen, we identified 14 independent clones that represented previously annotated proteins with a potential to regulate gene transcription (Table 1). Among these 14 clones, three clones containing fragments representing EPC1, DP1 and DP2 were identified [1,18,19,24]. Because these proteins have been shown to previously interact with E2Fs, this provided a strong validation of the screen. One additional clone contained a partial sequence coding for amino acids 462?78 of the BRG1 protein. Given that prior work has suggested a role for BRG1 in facilitating transcriptional regulation by a wide variety of proteins, we cloned full-length BRG1 and further confirmed its interaction with E2F6.E2F6 immunoprecipitates with BRGTo determine an interaction between BRG1 and E2F6, we first incubated S35-labeled in vitro translated BRG1 with an E2F6glutathionine S transferase (GST) fusion protein. Precipitation with GST beads revealed in vitro translated S35-labeled BRG1 associated with GST-E2F6 but not GST alone (Figure 1a). To confirm an interaction between E2F6 and BRG1 in cells, wecoexpressed E2F6 and BRG1 in T98G cells. E2F6 was shown to immunoprecipitate with BRG1 when an antibody recognizing BRG1 was used (Figure 1b). Given that we saw an interaction between BRG1, we also tested the ability of endogenous E2F6 and BRG1 proteins to interact under normal physiological conditions in another cell line. In agreement with our studies above, an antibody recognizing endogenous E2F6 was able to immunoprecipitate BRG1 in 293 cells (Figure 1C). To confirm the interaction between BRG1 and E2F6 was not resulting from a cross reaction between the antibodies recognizing E2F6 and BRG1, we determined an interaction between epitope tagged E2F6 and BRG1 proteins using antibodies to HA and Flag. An antibody recognizing HA tagged-E2F6 was able to immunoprecipitate Flag tagged-BRG1 only in cells that coexpressed HAE2F6 and Flag-Brg1 and not in cells expressing either protein alone (Figure 24195657 1D). We quantitated the blots by densitometry to obtain an approximation 
of the fraction of proteins bound (Table S1).E2F6 and BRG1 in Transcriptional RegulationFigure 2. BRG1 interacts with the E2F4 and E2F6. BRG1 specifically interacts with E2F4 and E2F6 but not with the activator E2Fs. A plasmid construct expressing BRG1 was cotransfected into 293 cells with plasmid constructs expressing HA-tagged E2F1, E2F2, E2F3, E2F4 or E2F6. Immunoprecipitation experiments were carried out in lysis buffer with a polyclonal antibody recognizing the HA epitope tag. Immunoprecipitates were resolved on SDS PAGE and Western blotted with a BRG1 monoclonal antibody. doi:10.1371/journal.pone.0047967.gBRG1 specifically interacts with E2F4 and E2FNumerous.
