Inhibition of NF-κB and IKK activity by NaAsO₂(A) HDLM-2 and L1236 cells were treated for 8 hours and L540 cells for 48 hours with the indicated concentrations of NaAsO₂. Whole cell extracts were analyzed by EMSA for NF-κB DNA binding activity. Free DNA probe is not shown; n.s. indicates nonspecific. (B) Expression level of IκBαmRNA and protein in response to NaAsO₂. After incubation of HDLM-2 and L1236 cells for 8 hours with the indicated concentrations of NaAsO₂, whole cell extracts were analyzed for IκBαprotein expression by Western blot analysis (WB, upper 2 blots). Total mRNA was prepared after 8 hours and analyzed for expression of IκBαand GAPDH by Northern blotting (NB, lower 4 blots). (C) Arsenite-mediated IKK inhibition. HDLM-2 cells were treated for 8 hours with PBS or 50 μM NaAsO₂, or for 48 hours with 1 to 5 μM NaAsO₂, as indicated. IKK activity was determined by an in vitro kinase assay (KA). IKKαwas immunoprecipitated using a monoclonal antibody against IKKαand protein A–Sepharose. The precipitates were incubated with IκBαand [γ-³²P]ATP at 37°C and subsequently analyzed by SDS-PAGE and autoradiography. As control, IKKαexpression was analyzed by Western blotting.