E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

Figure 6

E47 blocks PU.1 from binding to DNA in hematopoietic cells. (a) Immunoblot of PUER and E47 protein expression in nuclear extracts prepared from PUER GFP and PUER E47 cell lines which were grown in the absence or presence (+) of 100 nM OHT. (b) Electrophoretic mobility shift assay (EMSA) with a ³²P-labeled DNA probe from the MCSFR promoter. Nuclear extracts were prepared from PUER GFP and PUER E47 cell lines that were grown for 2 d in the absence or presence (+) of 100 nM OHT. SS indicates supershifted complex. (c) Chromatin immunoprecipitation (ChIP) of the MCSFR promoter from PUER GFP and 2 independent PUER E47 cell lines using anti-PU.1 antibody. PUER cell lines were treated for 7 d with OHT. Chromatin also precipitated with normal rabbit serum (NRS) to demonstrate specificity of the anti-PU.1 immunoprecipitations.

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