Figure 5.

RRM1 bound with hTrx1 by direct protein-protein interaction. A, SW480 cell lysates were co-immunoprecipitated with hTrx1 antibody and detected by immunoblotting. IgG was used as a negative control. B, SW480 cells were co-transfected with c-myc-RRM1 and FLAG-hTrx1 expression plasmids. The cell lysates were co-immunoprecipitated with FLAG antibody and detected by immunoblotting. C, SW480 cells were co-transfected with FLAG-RRM1 and c-myc-hTrx1 expression plasmids. The cell lysates were co-immunoprecipitated with FLAG antibody and detected by immunoblotting. Ten percent of each cell lysate for the co-immunoprecipitation was used for the input control. D, GST pulldown assay was performed by incubating purified RRM1-His protein with GST-hTrx1 immobilized on GST resin and analyzed by immunoblotting. E, GST pulldown assay was performed by incubating purified hTrx1-His protein with GST-RRM1 immobilized on GST resin and analyzed by immunoblotting. F, GST pulldown assay was performed by incubating purified RRM1-His or RRM2-His protein with GST-hTrx1 immobilized on GST resin at different time points in different buffers and analyzed by immunoblotting. G, SW480 cells were co-transfected with c-myc-RRM1 and si-RRM2. GST pulldown assay was performed by incubating the cell lysates with GST-hTrx1 immobilized on GST resin and analyzed by immunoblotting. H, SW480 cells were co-transfected with FLAG-RRM2 and si-RRM1. GST pulldown assay was performed by incubating the cell lysates with GST-hTrx1 immobilized on GST resin and analyzed by immunoblotting. IP, immunoprecipitation; ctr, control.