Fig 6.

Phosphorylation events in the IKK-CSN-complex. (A) Phosphorylation of Csn5/JAB1 by IKK2. Wild-type or mutant flag-IKK2 and flag-JAB1 were co-expressed in 293 cells, followed by immunoprecipitation with anti-flag beads and kinase assays as well as immunoblot analysis (anti-flag) of IKK2 and JAB1 expression. (B) Phosphorylation of c-Jun and IκBα by IKK2. Flag-tagged wild-type (wt) or mutant (mut) IKK2 was expressed in 293 cells, immunoprecipitated by anti-flag beads and kinase assays were performed as described in the ‘Materials and methods’ section using recombinant c-Jun, p53 (both from Biomol International, Inc., Hamburg, Germany) or GST-IκBα as substrates. A distinct phosphorylated band is visible for IκBα and a weaker band for c-Jun. (C) Phosphorylation of Csn5/ JAB1 by IKK1. 293 cells were transiently transfected with flag-tagged IKK1 and JAB1. MG132 was added to some samples to prevent proteasomal degradation. Anti-flag immunoprecipitates of cell extracts were subject to kinase assays and immunoblots against the flag-tag. JAB1-phosphorylation is most prominently visible in presence of MG132. (D) Auto-phosphorylation of IKK2 is enhanced by ectopically expressed JAB1. Flag-tagged wild-type or mutant IKK2 was coexpressed with flag-JAB1. Extracts were prepared 1 day after transfection and immunoblots were done for phospho-IKK2 (P-IKK2), flag and JAB1 as indicated. (E) Auto-phosphorylation of IKK1 is enhanced by ectopically expressed JAB1. Flag-tagged IKK1 and JAB1 were coexpressed and analysed as in (D). MG132 was added in one sample to block proteasomal degradation.