Inhibition of NF-κB signaling blocks CD40-mediated MM.1S migration, which is dependent on transcription and new protein synthesis.

(A) Serum-starved MM.1S cells were pretreated with either 10 μM PS1145 for 90 minutes or 1 μM SN50 peptides for 30 minutes, and then incubated with 2 μg/mL G28.5 mAb for the indicated intervals. Nuclear extracts from each sample were prepared, and oligonucleotides containing the consensus binding sequence of NF-κB bound to agarose beads were used to pull down nuclear NF-κB. The resulting samples were analyzed by Western blotting using an anti-p65 NF-κB Ab. (B) Serum-starved MM.1S cells were either left intact or pretreated with PS1145 and SN50 at the indicated concentration for 90 minutes and 30 minutes, respectively, and then added to the upper chamber in a transwell cluster plate for a transmigration assay. (C) MM.1S cells were transduced with Ad dnAKT, Ad myrAKT, or control Ad β-gal adenoviruses. Cells were preincubated with or without PS1145 (10 μM) and SN50 (1 μM), and a transmigration assay then performed triggered by G28.5 (5 μg/mL) anti-CD40 mAb. (D) Serum-starved cells were treated for 1 hour with act D or chx (1 and 10 μg/mL), and transmigration was then determined as described in “Materials and methods.” Data are the mean ±SD of triplicate determinations. Similar results were obtained in at least 2 additional experiments.