Figure 3 - MUC1 activates the IKK–IKK complex.

(a) HeLa-vector and HeLa-MUC1 cell lysates were separated on a Sephacryl S-200 HR column. The indicated fractions were analysed by immunoblotting with the indicated antibodies and for phosphorylation of GST–IB in kinase assays (KAs). (b) Anti-IKK immunoprecipitates from the indicated cells were immunoblotted with anti-IKK and anti-IKK antibodies. (c) GST or GST–IKK bound to glutathione beads was incubated with IKK in the absence and presence of MUC1-CD or MUC1-CD(mSRM). The precipitates were immunoblotted with an anti-IKK antibody (upper panel). Input of the proteins was assessed by immunoblotting with the indicated antibodies (lower 3 panels). (d) Anti-IKK precipitates from the indicated cells were immunoblotted with anti-phospho-IKK-Ser 181 and anti-IKK antibodies. (e) Anti-IKK precipitates from the indicated cells were incubated with GST-IB(1–54) and -32P-ATP. The reaction products were analysed by SDS–PAGE and autoradiography (upper panels). The precipitates were also immunoblotted with an anti-IKK antibody (lower panels). (f) GST or GST–IKK bound to glutathione beads was incubated with IKK in the absence and presence of MUC1-CD or MUC1-CD(mSRM). The precipitated complexes were suspended in kinase buffer containing ATP and incubated for 30 min at 30 °C. The reaction products were immunoblotted with an anti-phospho-IKK-Ser 181 antibody. Input of the GST–IKK, IKK and MUC1-CD proteins is shown in Fig. 3c. Full scans of the gels in a, b, c and f are shown in Supplementary Fig. S6-3.