GLI1 ubiquitination requires IKKβ and the E3 ligase ITCH. (A) SUDHL4 cells were stimulated with or without TNFα (20 μg/mL) for 15 minutes. Total cell lysates were subjected to anti-GLI1 immunoprecipitation. Bound proteins were detected with indicated antibodies. (B) FL-GLI1 was coexpressed in 293T cells in the presence or absence of ITCH construct. After 48 hours, cells were stimulated with or without TNFα for indicated time periods. Total cell lysates were subjected to anti-GLI1 immunoprecipitation, and bound proteins were detected with indicated antibodies. (C) Immunoprecipitation analysis of ubiquitinated-GLI1 in TNFα-stimulated 293T cells transfected with indicated constructs. (D) Immunoprecipitation analysis of K48 or K63 ubiquitinated-GLI1 in TNFα-stimulated 293T cells transfected with indicated constructs.