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. 2006 Jul 27;398(Pt 1):125–133. doi: 10.1042/BJ20060109

Figure 7. Treatment-specific interaction of SMC1 with OARRE or CAR.

Figure 7

(A) Nuclear extracts were prepared from the Ym17 cells treated with DMSO, TCPOBOP (250 nM), OA (10 nM) or TCPOBOP plus OA for 48 h and were applied to a DNA affinity column. After a wash with a buffer containing 0.3 M NaCl, nuclear proteins were eluted by 0.5 M NaCl from the (−257/−217)-beads or its mutated version (−257/−217mut)-beads and subjected to Western-blot analysis using SMC1 or V5 antibody. D, T, O and OT denote DMSO, TCPOBOP, OA and TCPOBOP plus OA respectively. Input is percentage of the proteins that were applied on columns. (B) Nuclear extracts prepared from DMSO- or OA-treated Ym17 cells were applied to the (−257/−217)-beads. SMC1 was precipitated from the flow-through (FT) or 0.3 M NaCl-washed fraction by incubation with 1 μg of SMC1 antibody (αSMC1) or normal rabbit IgG (nRb IgG). The resulting immunoprecipitates were resolved on 4–12% polyacrylamide gels and immunoblotted with the SMC1 or V5 antibody. Input is percentage of the proteins that were applied on columns. (C) ChIP assays to show the interaction of SMC1 with the promoter. Ym17 cells were treated with DMSO or TCPOBOP plus OA for 48 h, from which assays were performed for SMC1 antibody (αSMC1), V5 antibody (αV5), normal rabbit IgG (nRb IgG) or no antibody (no Ab). The purified DNA was amplified by PCR using primers specific to the −257/−217 region (amplicon −338 to −99) and resolved on a 1.5% agarose gel. Results shown were generated simultaneously from the same Ym17 cell extracts.