Fig. 1. MEKK3 is required for LPA-induced IKK-NF-κB activation.
(A) MEKK3+/+ and MEKK3-/- MEF cells reconstituted with empty vector or HA-MEKK3 were either untreated or treated with LPA (30 μM) for 0, 30, and 60 min, then harvested. Nuclear extracts were prepared and subjected to EMSA by using 32P-labeled NF-κB probes. Whole cell lysates (WCL) were subjected to SDS-PAGE and immunoblotting with antibodies indicated. PCNA was used as a loading control for nuclear extracts and β-actin was detected as a loading control for WCL. (B) MEKK3+/+ and MEKK3-/- MEF cells reconstituted with empty vector and MEKK3 were stimulated as in A. Then nuclear extracts were prepared and subjected to the immunoblotting analysis as indicated. (C) MEKK3-/- MEF cells reconstituted with empty vector and MEKK3 were either untreated or treated with LPA (30 μM) 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 LPA (30 μM) for 8 h. The relative luciferase activity was measured and normalized with the Renilla activity. Error bars indicate ± standard deviation in triplicate experiments.