Exposure of CML cells to bortezomib/flavopiridol induces perturbations in the IκB/NF-κB, SAPK, and MAPK pathways. (A) K562 cells were exposed to 8 nM bortezomib (Btzmb or Bz) ± 200 nM flavopiridol (FP) for 1 to 6 hours, after which the cells were lysed and subjected to Western blot analysis to monitor expression of phosphorylated IκBα. (B) Alternatively, K562 cells were treated for 24 hours as described for panel A, after which nuclear extracts were prepared and subjected to electrophoretic mobility shift assay (EMSA) as described in “Materials and methods.” For C + C′ (lane 1), 100-fold excess of unlabeled NF-κB oligonucleo-tides was preincubated for 10 minutes with the nuclear extract obtained from untreated cells prior to addition of labeled NF-κB oligonucleotides. Lane 6 was loaded with labeled NF-κB oligonucleotides. Results are representative of 3 separate experiments. (C) K562 (left) and LAMA84 (right) cells were incubated with Btzmb (K562: 8 nM; LAMA: 5 nM) ± FP (K562: 200 nM; LAMA: 150 nM) for 24 hours, after which cells were lysed and subjected to Western blot using the indicated antibodies. For panels A and C, each lane was loaded with 30 μg protein. An additional 2 studies yielded equivalent results.