FIG. 5.
PKRwt and the PKR mutants activate IKK. PKR0/0 cells were either untreated (None) or transfected with 5 μg of pcDNA1/Amp, alone (pc/Amp) or in the presence of 250 ng of pcDNA1/Amp expressing PKRwt or PKR/KR296. As a positive control, they were also treated for 30 min with 7 μg of LPS (Sigma) per ml or transfected with pCMV-Tax (Tax). Cell extracts (300 μg of protein/sample) were immunoprecipitated with anti-NEMO antibodies and analyzed for IKK activity in an in vitro phosphorylation assay with IκBα as a substrate (see Materials and Methods). The positions of phosphorylated IκBα (IκB-α-P) and IKKβ immunoprecipitated from the different cell extracts and revealed by immunoblotting are indicated.