IKKβ associates with GLI1 transcription factor. (A) To identify GLI1-interacting partners, we cotransfected full-length GLI1 construct in 293T cells, and the GLI1 complex was purified using anti-GLI1 antibody. Peptides resulting from digestion of GLI1 with proteases were analyzed by mass spectrometry. Identified proteins are shown in color according to their biological function. Red color depicts cell-cell signaling; purple, green, and blue colors depict signal transduction, protein biogenesis, and cell cycle, respectively. Endogenous immunoprecipitation of (B) GLI1 or (C) IKKβ in 293T and SUDHL4 cells. (D) GST-tagged IKKβ and affinity-purified GLI1 were used for an in vitro pull-down assay. (E) IKKα and IKKβ constructs were coexpressed in 293T cells with vector or full-length flag-tagged (FL)-GLI1, and total cell lysates were subjected to anti-GLI1 immunoprecipitation. (F) FL-GLI1 construct was coexpressed with vector, IKKβ, or IKKβ-KD (K44A) in 293T cells, and total cell lysates were subjected to anti-GLI1 immunoprecipitation. (G) IKKβ or IKKβ-KD was coexpressed with vector or FL-GLI1 in 293T cells, and total cell lysates were subjected to anti-IKKβ immunoprecipitation. Phosphorylation of IKKβ (Ser-181) confirmed activation of IKKβ in IKKβ-transfected cells. Red arrows depict slow-migratory GLI1 protein bands.