Fig 2.

IKKε phosphorylates TRAF2 at Ser11. (A) IKKε recognition motif and candidate TRAF2 sequences that match the motif. Highlighted residues (red) indicate a match to the recognition motif. Amino acid residues indicated as one-letter symbols (X, degenerate amino acid). (B) IKKε phosphorylates TRAF2 at Ser11. HEK293T cells were transfected as indicated. Myc-TRAF2 immune complexes were isolated and analyzed with antibodies specific for TRAF2, IKKε, and IKKε phosphosubstrates. (C and D) Effects of suppressing IKKε on TRAF2 Ser11 phosphorylation. Immunoblot of phospho-TRAF2 (Ser11) in HA1EM cells (C) or breast cancer cell lines (D) transduced with shLacZ or shLuc controls or IKKε-specific shRNAs (shIKKε#3 and shIKKε#5). Total IKKε, TRAF2, and β-actin expression is shown. The results of densitometry analysis are shown in the lower panel. (E) TRAF2 Ser11 phosphorylation in IKKε-transformed HA1EM cells. Immunoblotting was performed as described for panels C and D on HA1EM cells expressing a control vector, F-IKKε, or MF-IKKε. The results of densitometry analysis are shown in the lower panel.