Regulation of NF-κB distribution and activity by 5F11 and bortezomib. Detection of the NF-κB subunit p65 with anti-p65 and a secondary FITC-labeled antibody (green) in untreated L540 cells (A), after stimulation with cross-linked 5F11 for 6 and 8 hours (B-C), and after stimulation with bortezomib (D-E). The cell nuclei are stained with 4′6-diamidino-2-phenylindole (DAPI). Imaging medium was Vectashield (Vector Laboratories, Burlingame, CA). Pictures were taken using the digital Nikon Eclipse E800 microscope with the LuciaG/F program (Nikon, Düsseldorf, Germany). (A) 10 ×/0.17 NA objective; (B-E) 40 ×/0.11-0.23 NA objective. (F) L428 cells were transfected with either reporter construct pCMV-luc (transfection control) or pNF-κB-luc (bars 2-4) or pNF-κB-luc together with the expression vector IkBαM (bars 5-7) and exposed to PBS (basal), 5F11, or bortezomib for 24 hours. The luciferase activity (relative light units [RLU]) of the cell lysates is indicated. Error bars indicate mean and SD of 2 independent experiments. (G) EMSA to detect DNA binding of NF-κB in nuclear extracts of L540 and L428 cells after 6-hour incubation with no antibody, cross-linked 5F11, and cross-linked Ber-H2 (BH2) antibody. Ber-H2 as a control anti-CD30 antibody is unable to mediate CD30 signaling¹⁰  and fails to activate NF-κB. (H) Western blotting of whole L540 cell extracts to detect c-flip, bcl-2, and bax in untreated cells after 16-hour incubation (n.t. indicates no treatment) and following treatment with either 5F11 or 5F11 + bortezomib after 6-, 8-, and 16-hour incubation.