Fig 2: Phosphorylation of TAX1BP1 is required for localization to lysosomes. (A) Immunofluorescence assays of TAX1BP1 KO HeLa cells transfected with Flag-TAX1BP1 WT, 10A or 3SD (S254D, S593D and S666D) and 24 h later transfected with 2.5 µg/ml poly(I:C) for 6 h in the presence of 20 µm leupeptin. Scale bar: 10 µm. (B) Pearson’s correlation coefficient was calculated to measure colocalization between TAX1BP1 and LAMP1 in 8-12 cells randomly selected from each sample. Unpaired Student’s t-test, ***p < 0.001, n.s. = not significant.

Fig 3: TAX1BP1 degradation induced by poly(I:C), but not VSV infection, requires canonical macroautophagy/ATG8-family protein conjugation machinery. (A) Immunoblot analysis of control or ATG3 KO DLD-1 cells transfected with poly(I:C) at the indicated concentrations for 6 h. A lipofectamine only vehicle control is indicated as “LO.” (B) Immunoblot analysis of control or ATG3 KO DLD-1 cells infected with VSV-GFP at the indicated MOIs for 16 h. Densitometric analysis was performed using ImageJ.

Fig 4: TBK1 and IKBKE/IKKi regulate the basal turnover of TAX1BP1. (A) TAX1BP1 protein expression was quantified by ImageJ using lysates from WT and TBK1 IKBKE/IKKi dKO DLD-1 cells by normalizing TAX1BP1 expression to loading controls. Data were derived from eight independent experiments. Unpaired Student’s t-test, **p < 0.01. (B) Immunoblot analysis of extracts from DLD-1 cells treated with DMSO or Amlexanox (100 µg/ml) for 17 h. TAX1BP1 expression was quantified by ImageJ using lysates from DMSO- or Amlexanox-treated DLD-1 cells. Data were derived from nine independent experiments. Unpaired Student’s t-test, *p < 0.05. (C) WT and TBK1 IKBKE/IKKi dKO DLD-1 cells were treated with calyculin A for 30 min, and lysates were immunoblotted with the indicated antibodies. TAX1BP1 expression was quantified by ImageJ.

Fig 5: IκB kinases induce TAX1BP1 phosphorylation. (A) 293T cells were co-transfected with Flag-TAX1BP1 together with the indicated kinase plasmids and 24 h later lysed, and the cell extracts were immunoblotted with the indicated antibodies. (B) 293T cells were transfected with the indicated plasmids and lysates were incubated with λ-phosphatase for 30 min prior to immunoblotting with anti-Flag. (C) 293T cells were transfected with Flag-TAX1BP1 and either Flag-TBK1, Flag-IKBKE/IKKi or kinase dead mutants Flag-TBK1K38A or Flag-IKBKE/IKKiK38A, respectively. Lysates were subjected to immunoblotting with anti-Flag antibody. (D) in vitro kinase assays. Purified GST-tagged TAX1BP1 (300 ng) was incubated with 50 ng of purified recombinant GST-tagged IKBKE/IKKi or hexahistidine-tagged TBK1 in the presence or absence of ATP. The reaction mixtures were subjected to immunoblotting with antibodies to TAX1BP1, IKBKE/IKKi and TBK1. (E) DLD-1 cells were transfected with the indicated amount of plasmids for 24 h and then treated with BafA1 (20 nM) for 18 h. Cells were then lysed and subjected to immunoblotting analysis with the indicated antibodies. (F) Control and ATG3 KO DLD-1 cells were transfected with the indicated amount of plasmids for 24 h and then lysed and subjected to immunoblotting with the indicated antibodies. (G) Colloidal blue staining of in vitro kinase reaction mixtures containing TAX1BP1 with or without IKBKE/IKKi. Individual bands within the red rectangle were cut and gelextracted for mass spectrometry (MS) analysis. (H) Schematic diagram of TAX1BP1 domains. The indicated ten and thirteen predicted phosphorylation sites were substituted with alanine, generating the TAX1BP1 10A and 13A mutants, respectively. SKICH, the SKIP carboxy homology domain; LIR, LC3-interacting region; CC, coiled-coil domain; ZF, zinc finger domain.