Fig. 4: Stub1 promotes the degradation of TBK1.
A HEK293T cells were transfected with increasing amounts (0, 0.5, 1, and 2 µg) of a plasmid expressing HA-Stub1, and the protein was harvested for immunoblot analysis. B, C HEK293T cells were transfected with Flag-TBK1, together with increasing amounts (wedge) of HA-Stub1 expression vector, and the protein was harvested for immunoblot analysis. Flag-TBK1 expression was normalized to that of GAPDH (below) (B). C Right, RT-PCR analysis of TBK1 mRNA; GAPDH mRNA served as a loading control. D Immunoblot analysis of protein extracts from HEK293T cells transfected with Flag-TBK1 or HA-Stub1 and treated with cycloheximide (CHX) (100 μg/mL) for the indicated durations (0, 4, 8, or 12 h). Flag-TBK1 expression was normalized to that of GAPDH (below). E Protein lysates of HEK293T-stub1+/+ and HEK293T-stub1−/− cells infected with VSV (MOI = 0.1) at the indicated time points were immunoblotted with the indicated antibodies. F, G Immunoblot analysis of protein extracts from HEK293T-stub1+/+ and HEK293T-stub1−/− cells treated with cycloheximide (CHX) (100 μg/mL) for the indicated durations. H Immunoblot analysis of protein lysates of HEK293T cells transfected with vectors expressing Myc-Stub1-WT, Myc-Stub-K31A, Myc-Stub-H261A, or the Myc-Stub1-DM mutant, together with plasmids encoding Flag-TBK1. I Analysis of the Luc activity of the IFN-β promoter reporter in HEK293T cells transfected with vectors expressing Myc-Stub1-WT, Myc-Stub-K31A, Myc-Stub-H261A, or the Myc-Stub1-DM mutant together with plasmids encoding Flag-TBK1. J HEK293T cells were transfected with a plasmid encoding Flag-TBK1 together with plasmids encoding EV or Myc-Stub1, followed by treatment with MG132 (10 μM), 3MA (10 mM), CQ (50 μM), or bafilomycin A1 (Baf A1) (0.2 μM) for 6 h. The cell lysates were then analyzed by immunoblotting. Flag-TBK1 expression was normalized to that of GAPDH (below). K Protein lysates of WT, BECLN1 KO, ATG5 KO, and ATG7 KO 293T cells transfected with plasmids encoding EV or HA-Stub1. The data represent three independent experiments with three biological replicates (means ± SDs) or three independent experiments with similar results (A–K). Statistical significance was determined by two-tailed Student’s t test or one-way ANOVA followed by the Bonferroni post hoc correction, as appropriate. *P < 0.05, **P < 0.01, ***P < 0.001.
