KuAg is bound to HREs in gel mobility assays with nuclear extracts from hemin-treated U937 cells. (A) Supershift experiment. Gel mobility assays with radiolabeled 27-bp HRE probes were performed as described in Materials and Methods. Supershift experiments included an additional 30-minute incubation with Ab-3. Lane 1, standard gel mobility assay; lane 2, parallel supershift experiment with Ab-3. The arrow indicates the supershifted band. (B) Shift-Western experiments. Gel mobility assays with HRE probes were performed, followed by electrophoretic transfer of proteins and probes to stacked nitrocellulose and PVDF membranes as described in Materials and Methods. Panel 1, autoradiography of PVDF membrane. Remaining panels are immunoblots of nitrocellulose membrane with the anti-KuAg monoclonals Ab-4 (anti-P70), panel 2; and Ab-2 (anti-p80), panel 3; or with the anti-ref1 antibody, panel 4. Each panel depicts duplicate lanes. The arrow indicates the location of the shift band. The band at the top of each lane corresponds to the position of the sample wells. (C) Gel mobility assays with Ku depleted extracts. Nuclear extracts were prepared from hemin-treated U937 cells, then depleted of KuAg by serial incubations with Ab-3–derivatized magnetic beads as described in Materials and Methods. Panel I depicts standard gel mobility assays with radiolabeled 27-bp HRE probe. Lane 1, free probe; lane 2, undepleted extract; lanes 3 to 6 extract after 1, 2, 3, or 4 incubations, respectively, with anti-KuAg beads. Panel II: immunoblot of KuAg depleted extracts using antibody Ab-3. Lanes correspond to those of panel I. The arrow indicates the position of the largest gel shift band.