Maf1 associates with pol III, the individual pol III subunits RPC1 and RPAC2, and Brf1.

<p>A) Association of endogenous Maf1 with pol III and Brf1. Whole cell extract derived from the HeLa cell line 9–8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000134#pone.0000134-Hu1" target="_blank">[41]</a> was used for immunoprecipitations with anti-Maf1 or anti-GAPDH (Abcam) antibodies. The beads were washed, bound material was then eluted by boiling and used for immunoblotting with an anti-RPC1 antibody (CS377) (upper panel), an anti-Brf1 antibody (CS146) (middle panel) or an anti-Maf1 antibody (SZ2793) (lower panel). Lane 1 shows 1/20 of the input material, lanes 2, 3, and 4 show 1/30 of the flow through, 1/30 of the last wash, and 1/5 of the eluted material, respectively. Quantification of the signal shows that 6% of the RPC1 protein was co-immunoprecipitated with Maf1. B) Association of Maf1 with individual pol III subunits. GST-Maf1 or just GST were expressed in E. coli and immobilized on glutathione sepharose beads. Pol III subunits were synthesized by coupled <i>in vitro</i> transcription/translation in the presence of [³⁵S] methionine, and incubated with GST-Maf1 or GST beads. The beads were washed extensively and the bound material was analysed by SDS–PAGE and autoradiography. The first lane (input) shows 1/10 of the <i>in vitro</i> translated material added to the beads. C) Association of Maf1 with RPC1 and RPAC2 is not mediated by RNA nor DNA. Before the binding reaction, GST, GST-Maf1, and <i>in vitro</i> translated RPC1 and RPAC2 were subjected to RNase A or DNase I treatment as indicated above the lanes. D and E) Association of Maf1 with TFIIIB components and SNAP_c subunits, respectively. The experiments were performed as in B but with the <i>in vitro</i> translated proteins indicated. F) Pol III subunits and Brf1 associate with different Maf1 regions. On top, a schematic representation of full-length and truncated versions of human Maf1 are shown, with the A, B, and C conserved regions indicated. The experiment shown in the three bottom panels was performed as in B but with the GST fusion proteins indicated and <i>in vitro</i> translated GST, GST-Maf1^1-82, or GST-Maf1^1-142, as indicated.