GLI1 is a bona fide substrate of IKKβ. (A) 293T cells were transiently cotransfected with constructs carrying GLI1 and IKKβ wild-type. Cell lysates were incubated with or without calf intestinal alkaline phosphatase (CIP) and subjected to immunoblotting. The red arrow indicates phosphorylated GLI1 protein band. (B) Epitope-tagged expression vectors IKKβ or IKKβ-KD constructs were transfected into 293T cells. Immunoprecipitated kinase was assayed with affinity-purified GLI1 as a substrate in the presence of (γ³²P) ATP. (C) To identify IKKβ-dependent GLI1 phosphorylation sites, we cotransfected FL-GLI1 and IKKβ constructs in 293T cells, and the GLI1-complex was purified using anti-GLI1 antibody. Purified samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and band representing GLI protein was cut and analyzed by nanospray ion trap mass spectrometry. Eight phosphorylation sites were identified in the C-terminal region of GLI1 (S543-548, S1071, and T1074) as shown in the schematic diagram. (D) Immunoblot analysis of phosphorylated GLI1 in 293T cells transiently transfected with indicated GLI1 mutant constructs and IKKβ. (E) GST-purified GLI1 fragment (GLI1F; 411aa-1106aa) or triple-mutant GLI1 fragment (GLI1F^TM) protein was incubated with immunoprecipitated IKKβ kinase in the presence of (γ³²P) ATP and analyzed by in vitro kinase assay. (F) Cycloheximide (CHX) chase analysis of GLI1 or indicated full-length GLI1^TM construct was expressed in 293T cells in the presence or absence of CHX (60 μg/mL) for indicated time periods. After TNFα (20 μg/mL) stimulation, cell lysates from each time point were immunoblotted with anti-GLI1, anti-P-p65 (Ser536), and anti-p65 antibodies. (G) GLI1 luciferase reporter analysis of 293T cells transiently transfected with indicated constructs with or without TNFα stimulation for 6 hours. Results are normalized to Renilla luciferase and expressed as fold change in relative luciferase activity compared with control. Data represent the mean and standard deviation of 2 independent experiments (**P < .005). (H) FL-GLI1^TM construct was transiently transfected in 293T cells. After 48 hours, cells were stimulated with TNFα (20 μg/mL) for 60 minutes after CHX (60 μg/mL) and MG132 (30 μg/mL) treatment of indicated time periods. Cell lysates from each time point were immunoblotted with anti-GLI1 antibody. (I) 293T cells were cotransfected with full-length GLI1 and GLI1^TM mutant construct, and ITCH-IKKβ-GLI1 complex was purified using anti-IKKβ antibody and subjected to immunoblotting. Each component of GLI1 complex immunoblots were analyzed by densitometry, normalized with IKKβ, and plotted to determine normalized ITCH/GLI1 association.