Cytoplasmic NF- κ B levels are lowered by irinotecan but sorafenib reduces this effect. BT12 cells were incubated with sorafenib or vehicle for 30 minutes followed by treatment with irinotecan for an additional 2 hours. For indirect immunofluorescence (A), cells were fixed and stained with antibodies to NF-κ B followed by fluorescent labeled secondary antibodies. Concurrent DAPI stain was used to localize the nuclei (lower panel). Slides were visualized using a fluorescent microscope and random fields were photographed. The cytoplasmic staining seen in untreated and sorafenib treated cells was significantly reduced following treatment with irinotecan. However, the addition of sorafenib enabled the cells to maintain cytoplasmic staining in the presence of irinotecan. For Western blot analysis (B), cytoplasmic proteins were analyzed using antibodies against NFκ B p65 and p50, phospho-NFκ B p65, Iκ Bα and p27Kip1. In the presence of irinotecan, there was a loss of cytoplasmic NF-κ Bp65, but in the presence of sorafenib, this loss was greatly reduced, corresponding to a decrease in phosphorylation of NF-κ Bp65. In addition, compared to treatment with sorafenib or irinotecan alone, there was increased expression of Iκ Bα following treatment with sorafenib and irinotecan. Lastly, following treatment with irinotecan and sorafenib irinotecan combination, there was decreased expression of p27Kip1 compared to sorafenib treatment alone.