Fig. 2. MEKK3 is required for PKC-induced IKK/NF-κB activation.
(A) MEKK3+/+ and MEKK3-/- MEF cells reconstituted with empty vector and MEKK3 were either untreated or treated with PMA (40 ng/ml) plus Iono (100 ng/ml) for 0, 30, and 60 min. Nuclear extracts were prepared and subjected to EMSA by using 32P-labeled NF-κB probes. (B) MEKK3+/+ and MEKK3-/- MEF cells reconstituted with empty vector and MEKK3 were stimulated as in A. WCL and nuclear extracts isolated from the cells were subjected to SDS-PAGE and immunoblotting analysis as indicated. (C) MEKK3-/- MEF cells reconstituted with empty vector and MEKK3 were either untreated or treated with PMA (40 ng/ml) plus Iono (100 ng/ml) for 0, 5, 15, 30, and 60 min, then harvested. WCL were subjected to SDS-PAGE and immunoblotting analysis as indicated. (D) One µg of NF-κB luciferase reporter and 20 ng of Renilla-Luc plasmids were cotransfected into MEF cells indicated. Twenty-four hours after transfection, cells were starved for 12 h followed by the addition of PMA (40 ng/ml) plus Iono (100 ng/ml) for 8 h. The relative luciferase activity was measured and normalized with the Renilla activity. Error bars indicate ± standard deviation in triplicate experiments. (E) IKKβ+/+ and IKKβ-/- MEF cells were either untreated or treated with PMA (40 ng/ml) plus Iono (100 ng/ml) for 0, 30, and 60 min, then harvested. WCL and nuclear extracts were subjected to SDS-PAGE and immunoblotting analysis as indicated. (F) One μg of NF-κB luciferase reporter and 20 ng of Renilla-Luc plasmids were cotransfected into MEF cells indicated. Twenty-four hours after transfection, cells were starved for 12 h followed by the addition of PMA (40 ng/ml) plus Iono (100 ng/ml) for 8 h. The relative luciferase activity was measured and normalized with the Renilla activity. Error bars indicate ± standard deviation in triplicate experiments.