Bortezomib-resistant constitutive NF-κB activity in Rec-1 cells. (A) Chymotrypsin-like activity of the 26S proteasome in cell lines after exposure to increasing doses of bortezomib for 4 hours was assessed by luminescence generated by substrate cleavage (Proteasome-Glo Assay, Promega Corporation, Madison, WI). Results (mean ± 1SD from triplicate wells) are shown as a percent of luminescence relative to vehicle treated controls. (B) EMSAs of MCL cell lines treated with 20 nM bortezomib for 1 to 4 hours probed with 32P-radiolabeled oligonucleotide containing either the consensus NF-κB or Oct-1 binding sequence. Fold intensity was calculated by ImageQuant analysis of Phosphor Screens normalized to Oct-1 values from the same sample and then to the vehicle treated control values at the indicated time point. Gels shown are representative of one of three independent experiments. (C) EMSA of Rec-1 cells at time zero (0T) and treated with 0.1% DMSO (DMSO) for 3 hours, the calcium chelators BAPTA/AM (60 μM) and EGTA/AM (60 μM) for 3 hours, or the calmodulin inhibitors W12 (40 μM) and W13 (10 μM) for 3 hours. Fold activity refers to NF-κB binding normalized to Oct-1 binding in each condition. Gel shown is representative of 3 independent experiments.