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. 2012 Jun 18;7(6):e34692. doi: 10.1371/journal.pone.0034692

Figure 5. Chromatin immunoprecipitation (ChIP) assay demonstrated that expression of CARM1 resulted in reduced chromatin association of NuRD and TIF1β.

Figure 5

(A) Expression of CARM1 facilitates transcriptional activation by AR. HeLa cells were transfected with 100 ng pREP7-MMTV-Luc reporter, 20 ng pSG5-Flag-AR plus 0, 100 ng, 200 ng or 400 ng of pSG5-CARM1 and treated with or without 10 nM R1881 overnight. The relative luciferase activities were based on triplicates of two experiments. (B) Expression of CARM1 alone activates transcription from pREP7-MMTV-Luc reporter. pREP7-MMTV-Luc reporter was cotransfected with an increasing amount of pSG5-Flag-CARM1 plasmid. The relative luciferase activities from two independent experiments were shown. (C) ChIP analysis revealed that expression of CARM1 resulted in reduced association of NuRD and TIF1β with chromatin. HeLa cells were transfected with pREP7-MMTV-Luc reporter alone or plus 300 ng pSG5-Flag-CARM1. One day after transfection, the cells were collected and proceeded for ChIP analysis using antibodies as indicated. Left panel shows Western blot data for expression of Flag-CARM1. Arrow marks a non-specific band. PCR reactions were performed using a pair of MMTV-LTR specific primers and the products were revealed by agarose gel electrophoresis. (D) ChIP products in C) were analyzed by quantitative PCR.