Expression of proteins involved in canonical and noncanonical pathways modulating NF-κB activity in MM cell lines. (A) Nuclear extracts from MM cell lines were subjected to EMSA to assess baseline NF-κB activity. Lane 1 indicates MM.1S; lane 2, MM.1R; lane 3, U266; lane 4, INA6; lane 5, H929; lane 6, RPMI8226; lane 7, RPMI-LR5; lane 8, OPM1; and lane 9, OPM2. (B) Immunoblot analysis of IKKs, Rel family member, p50, and p52 proteins in MM cell lines (lanes 1-9 as noted in legend for panel A). Whole-cell lysates (W.L.) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by Western blotting with IKKα, IKKβ, IκB, and GAPDH Abs. Nuclear protein lysates (N.L.) were blotted with p50, p52, p65, RelB, cRel, and p84 nuclear protein Abs. Immunoblots are representative of similar results from 3 experiments. (C) To determine the role of Rel family proteins mediating constitutive NF-κB activity in MM.1S, U266, H929, RPMI8226, and OPM1 cells, supershift assays were carried out using anti-p65,(65), p50(50), p52(52), RelB (B), and c-Rel (cR) Abs. Exposure time of autoradiography varied for each cell line.