TRIM32 promotes TAX1BP1-mediated selective autophagic degradation of TRIF.

(A) Effects of ULK1/2-deficiency on poly(I:C)- or LPS-induced degradation of TRIF. Ulk1/2^+/+ and Ulk1/2^-/- MEFs were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (50 ng/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (B) Examination of the knockdown efficiency of FIP200-RNAi and ATG13-RNAi. HEK293-TLR3 cells (4 × 10⁵) were transfected with the indicated RNAi plasmids (1 μg each) for 36 hours before qPCR was performed. (C) Effects of FIP200-RNAi and ATG13-RNAi on poly(I:C)-induced degradation of TRIF. HEK293-TLR3 cells (4 × 10⁵) were transfected with the indicated RNAi plasmids (1 μg each) for 36 hours and then further treated with poly(I:C) (50 μg/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (D) Effects of NDP52 on levels of TRIF and TRAF6. HEK293 cells were transfected with the indicated plasmids before immunoblotting analysis with the indicated antibodies. (E) Interaction of TRIF with NDP52, TAX1BP1, OPTN and p62 in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 24 hours before communoprecipitation and immunoblotting analysis with the indicated antibodies. (F) Effects of TAX1BP1, OPTN and p62 on expression of TRIF. HEK293 cells were transfected with the indicated plasmids for 24 hours before immunoblotting analysis with the indicated antibodies. (G) Effects of TAX1BP1-RNAi on TRIM32-mediated degradation of TRIF. The control and TAX1BP1-RNAi stably transduced cells were transfected with the indicated plasmids for 24 hours before immunoblotting analysis with the indicated antibodies. (H) Effects of NDP52-RNAi or TAX1BP1-RNAi on poly(I:C)-induced degradation of TRIF. MLFs stably transduced with the indicated RNAi plasmids were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (I) Effects of TAX1BP1-RNAi on poly(I:C)-induced association of TRIF with LC3B. MLFs stably transduced with the indicated RNAi plasmids were left un-treated or treated with poly(I:C) (50 μg/ml) for the indicated times before coimmunoprecipitation was performed. (J) Effects of TAX1BP1-RNAi on poly(I:C)-induced transcription of Ifnb1, Cxcl10 and IL-6 genes. MLFs stably transduced with the indicated RNAi plasmids were left untreated or treated with poly(I:C) (50 μg/ml) for 2 hours before qPCR analysis. **, p<0.01.