Effects of TNF-α and a dominant-negative form of IKK-2 on FANCA phosphorylation.
GM6914 cells were transiently transfected with wt-FANCA, HA-IKK2(Lys44Ala) and NF-κB–luciferase reporter genes. At 48 hours after transfection, cells were used for further analyses. Whole cell extracts were used to confirm expression of HA-IKK2(Lys44Ala) by Western blotting with anti-HA monoclonal antibody. Cells were stimulated with TNF-α for 5 hours and cell lysates were prepared for assay of NF-κB activation. The values shown are averages ± SEM of triplicate samples for one representative experiment. To examine in vitro phosphorylation, lysates from cells stimulated with TNF-α (20 ng/mL) for 5 minutes were subjected to immunoprecipitation with anti-FANCA antibody. To examine in vivo phosphorylation, cells were metabolically labeled with 32P, stimulated with TNF-α (20 ng/mL) for 5 minutes and lysates were prepared. The lower panels show Western blots of FANCA.
