EF24 does not inhibit xFANCD2-Ub through modulation of a phosphorylation event, but IKK inhibition might play a role. A) EF24 inhibits the IKK kinase in Xenopus extracts. Extracts treated as indicated were analyzed by immunoblot using xFANCD2, IκB-α and tubulin-α antibodies. IκB-α protein level was used as readout for monitoring IKK inhibition. B) The specific IKK inhibitor compound BMS-345541 (IKK inhibitor III, Calbiochem) inhibits xFANCD2-Ub in extracts. Experiment was performed as in (A). The structure of BMS-345541 is shown. The star denotes a non-specific band used as loading control. C) EF24-dependent inhibition of xFANCD2-Ub is not affected by co-treatment with tautomycin (phosphatase inhibitor), caffeine (kinase inhibitor) and SAP (shrimp alkaline phosphatase). Extracts treated as indicated were analyzed by immunoblot using xFANCD2 and xMRE11 antibodies. xMRE11 phosphorylation status was used to monitor the efficiency of tautomycin, caffeine and SAP treatments.