Fig. 4

Morgana is an essential component of the IKK complex in cancer cells. a, b Western blot of MDA-MB-231 a and BT549 b infected with an empty vector (EMPTY) or two Morgana shRNAs (shMORG1, shMORG2). c, d Immunoblot of MCF10A c and MCF7 d infected with an empty vector (EMPTY) or overexpressing Morgana (MORGANA). e, f Immunoprecipitation of Morgana from MDA-MB-231 e or BT549 f immunoblotted with IKKα, IKKβ, IKKγ, IκBα and Morgana. g Gel filtration analysis of the endogenous IKK complex purified from MDA-MB-231. The total protein extracts were fractionated on a Superose 6 gel filtration column. Each fraction was analyzed by Western blot. The arrows indicate the fractions in which Morgana and the IKK complex eluted together. h Immunoprecipitation of Morgana from selected fractions obtained by gel filtration immunochromatography of MDA-MB-231 immunoblotted with IKKα, IKKβ, HSP90 and Morgana. i Gel filtration analysis of total protein extracts from MDA-MB-231 shMORG1 (right) compared with control cells (left). Each fraction was analyzed by Western blot. The arrows indicate the fractions in which Morgana and the IKK complex eluted together. j Immunoprecipitation of IKKβ from MDA-MB-231 immunoblotted with IκBα, IKKα and IKKβ. k IKKβ was immunoprecipitated from MDA-MB-231 EMPTY and shMORG and subjected to an in vitro kinase assay with or without addiction of recombinant MBP-MORGANA or MBP as control. The IKKβ inhibitor, PS1145, was added to IKKβ immunoprecipitation from MDA-MB-231 EMPTY, as control. The graph shows the average intensity of the P-IκBα bands normalized to IκBα. l Immunoblotting of P-IκBα, IκBα, IKKβ, Morgana and Vinculin in MDA-MB-231 EMPTY or shMORG1 and shMORG2 transfected or not with IKKβ wild type (WT) or constitutively active (CA). Data are the results of three independent experiments. Bars in graphs represent standard errors (*p < 0.05; **p < 0.01)