FIGURE 5.

AND-1 interacts with cohesin complex and is required for recombinational repair. A, nuclear extracts (700 μg of protein in top panel; 30 μg of proteins in bottom panel) of HeLa S3 cells were immunoprecipitated (IP) with anti-AND-1C antibody or preimmune serum (Pre) and subjected to immunoblot analysis. In the top panel, input (x5 in lane 1 and x1 in lane 2) represents 0.1 or 0.02%, respectively, of the starting extracts used for immunoprecipitation in lanes 3 and 4. In the bottom panel, input represents 20% of the starting extracts used in lane 3. The asterisk indicates a nonspecific signal. B, HeLa cells were transfected with pME18S-FLAG-AND-1 (+) (lanes 2 and 4) or pME18S (−) (lanes 1 and 3) and harvested at 48 h after transfection. Cells were lysed and immunoprecipitated (IP) with anti-FLAG M2-agarose and analyzed for co-immunoprecipitation of Smc1 protein. Input (lanes 1 and 2) represents 10 μg of the starting extracts used for immunoprecipitation in lanes 3 and 4, respectively. In B, contrast-enhanced images of long exposed films are also shown. C, analysis of I-SceI-induced homologous recombinational repair using a neomycin-based reporter construct. The frequency of neomycin-resistant colonies was counted in control, AND-1 number 1, or AND-1 number 3 siRNA-transfected SW480sn3 cells. The error bars represent S.D. from three independent experiments, and p values are less than 0.01 for both AND-1 number 1 and AND-1 number 3 siRNAs compared with control. Values of cells transfected with I-SceI pCMV3his-I-SceI (gray bars) or empty vector (open bars) are shown. Similar defects in recombinational repair were observed also in cells transfected with AND-1 number 4 and number 5 siRNA (data not shown).