Skip to main content
Figure 4 | Journal of Experimental & Clinical Cancer Research

Figure 4

From: Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction

Figure 4

Chemical treatment promotes binding of TOPO1 and YB-1 through oxidative stress. A. Identification of the YB-1- TOPO1 interaction in Flag-YB-1 stably-transfected PC-3 cells. Whole-cell lysates (500 μg) prepared from two clones (cl35 and cl37) of PC-3 cells transfected with the pcDNA3-Flag-YB-1 expression plasmid were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-Flag (upper panel) or anti-YB-1 (lower panel) antibodies. Both Flag-YB-1 and endogenous YB-1 are shown (left panel). Nuclear extracts (500 μg) of control cells or cl37 PC-3 cells were immunoprecipitated with the anti-Flag (M2) antibody. The resulting immunocomplexes were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Transferred proteins were probed with the anti-TOPO1 antibody. The band labeled with an asterisk was identified as TOPO1. The Flag-YB-1 protein band is indicated by an arrow. B. Chemical treatment increases YB-1-TOPO1 complex. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated for 4 h with the concentrations of CPT or ADM indicated. Nuclear extracts were immunoprecipitated with the anti- Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. C. Longer CPT treatment increased YB-1-TOPO1 interaction and YB-1 expression. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated with the indicated concentrations of CPT for 4 h and 24 h respectively. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. D. Pretreatment with NAC prevented the chemically augmented YB-1-TOPO1 interaction. Cl37 of Flag-YB-1 stably-transfected PC-3 cells were untreated or preincubated with NAC (30 mM, 1 h) before addition of CPT. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies.

Back to article page