Inhibition of IKKβ does not block proliferation of MM cell lines which express both canonical and noncanonical NF-κB pathways. (A) MM.1S (○), MM.1R (■), U266 (□), INA6 (●), H929 (▴), RPMI8226 (♦), RPMI-LR5 (◇), OPM1 (Δ), and OPM2 (×) cells were cultured in the presence of MLN120B (0-40 μM) for 72 hours. Cell proliferation was assessed by 3H-thymidine uptake. (B) OPM1, MM.1S, and RPMI8226 cells were treated with or without MLN120B (5 μM) for the indicated time intervals. Nuclear extracts were subjected to EMSA to assess NF-κB activity. Exposure time of autoradiography varied for each cell line and intensity of the bands were digitalized by ImageJ software and indicated as fold-increase relative to control. (C) To determine the role of Rel family proteins mediating NF-κB activity in OPM1 cells before and after MLN120 treatment, supershift assays were carried out using anti-p65(65), p50(50), p52(52), RelB (R-B), and c-Rel (cR) Abs.
