D screen.Clone MIZ1 FAZF DNMT1 BRG1* NF-Yalpha BRAF35 BRDT ZFPM

D screen.Clone MIZ1 FAZF DNMT1 BRG1* NF-Yalpha BRAF35 BRDT ZFPM2 DNTTIP1 IN080B EPC1 KLIP1 DP1 DPGenbank Accession No. NM_003443 NM_014383 NM_001379 NM_003072 NM_021705 NM_006339 NM_001726 NM_012082 NM_052951 NM_031288 NM_025209 NM_024629 NM_026580 NM_E2F1 + + + 2 + + + + + + + + + +E2F2 2 2 2 2 2 2 2 2 2 2 2 2 + +E2F3 + + 2 2 + 2 2 + 2 2 + 2 + +E2F4 2 2 2 2 2 2 2 2 2 2 2 2 + +E2F6 + + + + + + + + + + + + + +Immunoprecipitates were eluted by boiling in 2 X Laemelli’s buffer and then separated by SDS-PAGE. Western blots were carried out with the desired antibodies. E2F6, BRG1, DP1, BAF155 and HA proteins in immunopreciptiations and Western blots were carried out using antibodies from Santa Cruz (E2F6, sc8175 sc-8366; HA, sc-805 sc-7392; BRG1, sc-17796 sc10768; DP1, sc-610 sc-53642; BAF155, sc-32763). The antibody recognizing the Flag epitope tag was purchased from Sigma (F3165). The antibody recognizing BAF180 was purchased from Millipore (ABE70). The density of bands on Western blots were quantitated using the publicly available software, Image J.Chromatin immunoprecipitation assaysChromatin immunoprecipitation assays were carried out as previously described [5,6,29,30,31]. Nuclear chromatin extracts were incubated with antibodies from Santa Cruz Biotechnology described above at 4uC overnight. Immunoprecipitates were collected on 25 ml Protein G agarose beads (Roche) for 1? h at 4uC. After thorough washing, immunoprecipitates were decrosslinked and chromatin was recovered on Qiagen miniprep spin columns. Quantitiative PCR (qPCR) analyses were performed in real time using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix following the manufacturer’s protocol. Relative occupancy values were calculated as described previously [31]. Final values reflecting promoter occupancy are reported as the percent of input DNA, immunoprecipitated from each antibody after adjusting to input levels and normalization to albumin levels. Primer sets for qPCR are available upon request.Clones identified from the yeast two-hybrid screen were tested for an interaction with E2Fs1? and E2F6 in yeast. Full length human E2Fs 1? and E2F6 were cloned into pGBT9B as described in Materials and MedChemExpress 47931-85-1 Methods and the resulting plasmid, along with DP1, was transformed into yeast to test for an interaction with the indicated proteins. *CASIN price Ectopic expression of E2F4 was able to immunoprecipitate ectopically expressed BRG1 in T98G cells even though an interaction was not observed in yeast. doi:10.1371/journal.pone.0047967.tLuciferase Reporter AssaysReporter assays to determine the effects of E2F6 on E2F1 promoter activity were carried out on 6-well dishes. Cells transfected 24 h after plating with 1 ug of the E2F1 reporter, 1 ug of a LacZ expressing plasmid and E2F6-pcDNA3 at concentrations of 0?0 ng per well. The experiment to determine the effects of a dominant BRG1 on E2F6-mediated repression was performed after transfection with 12926553 0.8 ug of a lac Z expressing plasmid, 0.8 ug of the E2F1 reporter, 250 ng of a plasmid expressing dominant negative BRG1 and 9 ng of E2F6. The total mass of transfected DNA in each well was kept constant by adding empty vector plasmid DNA, when necessary. All experiments were performed in triplicates, and mean 6s.d. values were determined. All transfections were performed overnight at 37uC and then cells were allowed to recover in 10 FBS MEM. At 18?6 h after transfection, cells were washed with 1 6 PBS and then lysed with 16.D screen.Clone MIZ1 FAZF DNMT1 BRG1* NF-Yalpha BRAF35 BRDT ZFPM2 DNTTIP1 IN080B EPC1 KLIP1 DP1 DPGenbank Accession No. NM_003443 NM_014383 NM_001379 NM_003072 NM_021705 NM_006339 NM_001726 NM_012082 NM_052951 NM_031288 NM_025209 NM_024629 NM_026580 NM_E2F1 + + + 2 + + + + + + + + + +E2F2 2 2 2 2 2 2 2 2 2 2 2 2 + +E2F3 + + 2 2 + 2 2 + 2 2 + 2 + +E2F4 2 2 2 2 2 2 2 2 2 2 2 2 + +E2F6 + + + + + + + + + + + + + +Immunoprecipitates were eluted by boiling in 2 X Laemelli’s buffer and then separated by SDS-PAGE. Western blots were carried out with the desired antibodies. E2F6, BRG1, DP1, BAF155 and HA proteins in immunopreciptiations and Western blots were carried out using antibodies from Santa Cruz (E2F6, sc8175 sc-8366; HA, sc-805 sc-7392; BRG1, sc-17796 sc10768; DP1, sc-610 sc-53642; BAF155, sc-32763). The antibody recognizing the Flag epitope tag was purchased from Sigma (F3165). The antibody recognizing BAF180 was purchased from Millipore (ABE70). The density of bands on Western blots were quantitated using the publicly available software, Image J.Chromatin immunoprecipitation assaysChromatin immunoprecipitation assays were carried out as previously described [5,6,29,30,31]. Nuclear chromatin extracts were incubated with antibodies from Santa Cruz Biotechnology described above at 4uC overnight. Immunoprecipitates were collected on 25 ml Protein G agarose beads (Roche) for 1? h at 4uC. After thorough washing, immunoprecipitates were decrosslinked and chromatin was recovered on Qiagen miniprep spin columns. Quantitiative PCR (qPCR) analyses were performed in real time using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix following the manufacturer’s protocol. Relative occupancy values were calculated as described previously [31]. Final values reflecting promoter occupancy are reported as the percent of input DNA, immunoprecipitated from each antibody after adjusting to input levels and normalization to albumin levels. Primer sets for qPCR are available upon request.Clones identified from the yeast two-hybrid screen were tested for an interaction with E2Fs1? and E2F6 in yeast. Full length human E2Fs 1? and E2F6 were cloned into pGBT9B as described in Materials and Methods and the resulting plasmid, along with DP1, was transformed into yeast to test for an interaction with the indicated proteins. *Ectopic expression of E2F4 was able to immunoprecipitate ectopically expressed BRG1 in T98G cells even though an interaction was not observed in yeast. doi:10.1371/journal.pone.0047967.tLuciferase Reporter AssaysReporter assays to determine the effects of E2F6 on E2F1 promoter activity were carried out on 6-well dishes. Cells transfected 24 h after plating with 1 ug of the E2F1 reporter, 1 ug of a LacZ expressing plasmid and E2F6-pcDNA3 at concentrations of 0?0 ng per well. The experiment to determine the effects of a dominant BRG1 on E2F6-mediated repression was performed after transfection with 12926553 0.8 ug of a lac Z expressing plasmid, 0.8 ug of the E2F1 reporter, 250 ng of a plasmid expressing dominant negative BRG1 and 9 ng of E2F6. The total mass of transfected DNA in each well was kept constant by adding empty vector plasmid DNA, when necessary. All experiments were performed in triplicates, and mean 6s.d. values were determined. All transfections were performed overnight at 37uC and then cells were allowed to recover in 10 FBS MEM. At 18?6 h after transfection, cells were washed with 1 6 PBS and then lysed with 16.

Leave a Reply