Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: Three phosphoserine residues are involved in TAX1BP1 degradation. (A) Immunoblotting analysis of the extracts derived from 293T cells transfected with WT Flag-TAX1BP1 and mutants (10A and 13A) together with, or without, Flag-IKBKE/IKKi. For better separation of phosphorylated TAX1BP1, a 6% gel was used. (B) Schematic of restored TAX1BP1 10A variants at single or multiple phosphorylation residue(s). (C) 293T cells transfected with WT Flag-TAX1BP1 and mutants together with HA-tagged TAX1BP1 10A for 24 h were infected with or without VSV-GFP for 6 h at an MOI of 1, and the cell extracts were subjected to immunoblotting with the indicated antibodies. (D) Densitometric analysis of TAX1BP1 band intensity from three independent experiments presented in (C). Densitometric analysis was performed with ImageJ. Unpaired Student’s t -test, ** p < 0.01, * p < 0.05. (E) Immunoblotting analysis of the extracts derived from 293T cells transfected with WT Flag-TAX1BP1 and variants (10A, 7A, and 6A) and 24 h later infected with or without VSV-GFP as above. As shown in (B), 7A was generated by restoring the three potential phosphorylation sites, S254, S593, and S666 in 10A, and 6A was generated by restoring T250 in 7A.

Article Snippet: ImageJ software (NIH) or Image Lab software (Bio-Rad) was used to quantify the intensities of bands.

Techniques: Western Blot, Derivative Assay, Transfection, Phospho-proteomics, Residue, Infection, Generated

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: TAX1BP1 is targeted to LC3-positive vesicles in response to poly(I:C) transfection. (A) Immunofluorescence assays of WT and TBK1 IKBKE/IKKi dKO DLD-1 cells transfected with 5 µg/ml poly(I:C) for 6 h in the presence of BafA1 (100 nM). Scale bar: 10 µm. (B) Colocalization analysis of TAX1BP1 and LC3 following poly(I:C) transfection. Manders coefficients were derived using ImageJ. Coefficients shown are representative of TAX1BP1 signal overlapping with LC3 signal from 50 cells chosen at random across five fields per condition. Unpaired Student’s t -test. **** p < 0.0001, * p < 0.05.

Article Snippet: ImageJ software (NIH) or Image Lab software (Bio-Rad) was used to quantify the intensities of bands.

Techniques: Transfection, Immunofluorescence, Derivative Assay

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: 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.

Article Snippet: ImageJ software (NIH) or Image Lab software (Bio-Rad) was used to quantify the intensities of bands.

Techniques: Expressing, Derivative Assay, Western Blot

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: 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.

Article Snippet: ImageJ software (NIH) or Image Lab software (Bio-Rad) was used to quantify the intensities of bands.

Techniques: Infection, Conjugation Assay, Western Blot, Control, Transfection

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: RB1CC1/FIP200 is required for TAX1BP1 degradation in response to VSV infection and poly(I:C) transfection. (A) WT, rb1cc1 –/– and atg3 –/– MEFs were infected with VSV-GFP (MOI = 1) for 13 h and subjected to immunoblotting with anti-TAX1BP1 and anti-ACTB/β-actin antibodies. (B) Control and RB1CC1/FIP200 KO DLD-1 cells were infected with VSV-GFP at the indicated MOIs for 20 h and then subjected to immunoblotting analysis with the indicated antibodies. Densitometric analysis was performed using ImageJ. (C) Control and RB1CC1/FIP200 KO DLD-1 cells were transfected with the indicated concentration of poly(I:C) for 6 h and then subjected to immunoblotting analysis with the indicated antibodies. A lipofectamine only control is designated as “LO.” Densitometric analysis was performed using ImageJ. (D) Control and RB1CC1/FIP200 KO cells stably expressing a TAX1BP1-eGFP fusion protein were infected with VSV for 20 h (MOI = 1) and then analyzed by flow cytometric analysis of eGFP fluorescence intensity. Geometric mean fluorescence intensity (gMFI) was quantified using FlowJo 10 software. Data were derived from three independent experiments. Unpaired Student’s t -test. ** p < 0.01.

Article Snippet: ImageJ software (NIH) or Image Lab software (Bio-Rad) was used to quantify the intensities of bands.

Techniques: Infection, Transfection, Western Blot, Control, Concentration Assay, Stable Transfection, Expressing, Fluorescence, Software, Derivative Assay