TNFα-mediated activation of IKKβ suppresses GLI1 ubiquitination. (A) Immunoprecipitation analysis of ubiquitinated-GLI1 in SUDHL4 cells stimulated with or without TNFα (20 μg/mL) for 15 minutes. (B) Indicated constructs were coexpressed in 293T cells. Before cell lysis, cells were stimulated with or without TNFα for indicated time periods. Total cell lysates were subjected to anti-GLI1 immunoprecipitation. Bound ubiquitinated-GLI1 was detected by anti-HA antibody. (C) Immunoprecipitation analysis of total ubiquitinated-GLI1 in control (Cont.) or partially IKKβ-depleted HBL1 cells after treated with proteasome inhibitor MG132 (10 μg/mL) for 6 hours. (D) Immunoprecipitation analysis of K48 or K63-linked ubiquitinated-GLI1 in 293T cells transfected with FL-GLI1 and HA-K48 or HA-K63–linked ubiquitin constructs. Before cell lysis, cells were stimulated with or without TNFα (20 μg/mL) for indicated time periods.