Figure 5

Co-recruitment of PNRC and RPC39 onto RNA polymerase III-dependent genes. MCF-7/EGFP-PNRC cells were transfected either with pArg maxi reporter plasmid, which contains tRNA^arg gene promoter and coding sequence, or with pTZU6 plasmid that carries the U6 RNA gene promoter. Twenty-four hours after transfection, cells were treated with formaldehyde to crosslink endogenous proteins and DNA. Samples of sonicated and purified chromatin were immunoprecipitated with no antibody (No ab), preimmuno IgG (IgG), GFP antibody (anti-GFP), or RPC39 antibody (anti-RPC) as indicated. DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify a 220 bp fragment of the Drosophila tRNA^arg gene (A) or a 160 bp fragment of the U6 RNA gene (B). The amplified PCR products were analyzed on 1.8% agarose gel. The PCR products from the reactions using input DNA, pArg or pTZU6 plasmids as template were included on the gels as positive and size controls.