Skip to main content
. 2000 Aug;20(15):5381–5391. doi: 10.1128/mcb.20.15.5381-5391.2000

FIG. 7.

FIG. 7

IKK activity mediates constitutive NF-κB activation in oncogenic Ras- or Raf-transformed RLEs. (A) Inhibition of IKK activity. Cells were transiently transfected in P60 dishes with 2 μg of E8 or dmE8 NF-κB CAT construct in the absence or presence of 2 μg of vector directing expression dnIKK form IKK-1 SS/AA or IKK-2 SS/AA (51). As controls, the parental pRC-βactin and pCMV-Neo constructs, respectively, were similarly transfected. The values for E8 CAT activity are represented as fold induction over dmE8 CAT activity, which was set at 1.0 for each cell line. (B) Mutant version of IKKAP1 inhibits NF-κB activation in F22-Ras cells. Cells in P60 dishes were transiently transfected with 2 μg of E8 or dmE8 NF-κB CAT construct in the absence or presence of 2 μg of DNA of a vector directing expression of either full-length (FL) IKKAP1 or its C-terminus-deleted version ΔC IKKAP1 (51). In addition, 1 μg of simian virus 40–β-Gal expression vector was added to normalize for transfection efficiency. As control, the parental pCDNA3-EE construct was similarly transfected. The values are represented as percentage relative to the CAT activity of the E8 reporter in the presence of the parental vector, which was set at 100%. Means and standard deviations are representative of two independent experiments carried out in duplicate. (C) Kinase assays. (Top) Cells were transiently transfected with 5 μg of vector directing expression of wt IKK-1 or IKK-2 as described above. Following immunoprecipitation with antibodies against IKK-1 or IKK-2, extracts (10 μg) were subjected to kinase assays using either wt IκB-α–GST or the Ser32/36 double-mutant IκB-α–GST version, which cannot be phosphorylated. (Bottom) Equal aliquots of the immunoprecipitates from the lanes in the top panel, as indicated, were subjected to immunoblotting for IKK-1 and IKK-2 protein. (D) Extracts from exponentially growing cells (80 μg) were immunoprecipitated with antibodies against IKK-1 or IKK-2. Immunoprecipitated proteins were subjected to kinase assay using wt IκB-α–GST as substrate.