Regulation of TNIP1 protein abundance. (A) Reduction of TNIP1 correlates with an increase of ISG15 and TNFAIP3/A20 in HeLa cells. The increase of the TNIP1 interaction partner TNFAIP3 under poly(I:C) treatment indicates the existence of distinct TNIP1 pools, i.e., free and bound to TNFAIP3. Arrow marks A20 band. (B) Poly(I:C) treatment leads to an autophagy-dependent and SLR-independent lysosomal degradation of TNIP1. Whereas lysosomal inhibition by ConA treatment leads to a significant block of TNIP1 degradation in WT and pentaKO cells, proteasomal inhibition by MG132 treatment has no effect. In ATG101 KO, FIP200 KO and ATG7 KO, autophagy incompetent cell lines poly(I:C) does not lead to TNIP1 degradation. TLR3 and SQSTM1 are monitored as positive controls, actin as loading control. Shown are representative blots of three biological replicates each. Bar diagram shows quantification, error bars: SEM. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, unpaired, two-sided t test. (C) TNIP1 KO cells transfected with an empty control vector (EV) do also respond to poly(I:C) treatment by an upregulation of ISG15 and CCL5 similar to KO cells transfected with a TNIP1 expression construct. This indicates that ISG15 and CCL5 abundances are not only regulated by TNIP1. Shown are representative blots of three biological replicates. Bar diagram shows quantification. Error bars represent SEM. * = P < 0.05, ** = P < 0.01, *** = P < 0.001; unpaired, two-sided t test. (D) Blockage of protein translation by cycloheximide (CHX) treatment reduced the time- and poly(I:C)-dependent increase of TNIP1 after 6 h of treatment indicating a regulation on translational level. Source data are available for this figure: SourceData FS4.