Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the pelleting assay. B) Immunoblotting for autophagy factors after Tau aggregation and the pelleting assay. C) Quantification of the signal in the pellet fraction in comparison to the input samples. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Multiple unpaired t-tests. D) Immunofluorescence images of Tau, p62, TAX1BP1, ATG13 and TBK1 p-S172 (scale bar = 20 µm). E) Pearson’s correlation (PCC) between Tau aggregates and p62, TAX1BP1, ATG13 and TBK1 p-S172. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to the measured correlation within an individual cell. One-way ANOVA with Dunnett’s multiple comparisons test. F) Immunofluorescence images for p62 and p62 p-S403 in cells expressing Tau-mRuby after Tau seeding (scale bar = 20 µm). G) Pearson’s correlation (PCC) between Tau aggregates and p62 or p62 p-S403 in I. Data are presented as violin plots showing the distribution of individual data points. The solid line indicates the median; dashed lines represent the interquartile range. Each data point corresponds to measured correlation within an individual cell. H) Schematic summary of the results. *P < 0.05, **P < 0.005, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Western Blot, Comparison, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of the domain structure of TAX1BP1 and the tethering of TAX1BP1 truncations. B) Representative fluorescence-activated cell sorting (FACS) plots of Tau flux measurements in cells expressing full-length TAX1BP1, TAX1BP1ΔSKICH or TAX1BP1ΔZnF. C) FACS-based measurement of Tau flux upon its aggregation and tethering of TAX1BP1 WT, TAX1BP1ΔSKICH and TAX1BP1ΔZnF. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. D) Schematic overview of the microscopy-based protein-protein interaction assay in E. E) Representative images of microscopy-based protein-protein interaction assays between the SKICH domain of TAX1BP1 on the beads and GFP-ULK1 complex, mCh-PI3K complex, mCh-WIPI2, GFP-TBK1 and mCh-NAP1 (scale bar = 80 µm). F) Schematic depiction of the formation of the initiation hub on the surface of the SKICH domain of TAX1BP1 together with a size comparison between the SKICH domain and the proteins involved in the formation of the complex. G) Immunofluorescence images of TAX1BP1, ATG13, ULK1, FIP200 and TBK1 p-S172 with and without addition of rapalog (scale bar = 20 µm). ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Fluorescence, FACS, Expressing, Microscopy, Protein Protein Interaction Assay, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Immunoblotting of phospho-ULK1, ATG13, TBK1 and p62 upon Tau aggregation and TAX1BP1 tethering. B) Quantification of relative increase of phosphorylation levels upon TAX1BP1 tethering. Phosphorylation signal was normalized to the total protein. To account for inter-experimental variability, values are expressed as a relative to the -Rapalog samples within each biological replicate. The data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Sidak’s multiple comparisons test. *P < 0.05, **P < 0.005. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: TAX1BP1 recruitment reactivates autophagy of Tau aggregates through ULK1 and TBK1

doi: 10.64898/2026.02.12.705532

Figure Lengend Snippet: A) Schematic overview of tethering ATG13 to Tau aggregates using the FKBP-FRB-Rapalog system. B) Immunofluorescence images of Tau and ATG13 (scale bar = 20 µm). C) Immunoblotting of ATG13, ULK1, FIP200 and TBK1 with and without addition of rapalog. D) Quantification of the fraction of protein pelleted upon rapalog treatment in C. E) Immunofluorescence images of Tau and ULK1 or FIP200 with and without rapalog treatment (scale bar = 20 µm) F) Schematic overview of tethering TBK1 to Tau aggregates using the FKBP-FRB-Rapalog system. G) Immunofluorescence images of Tau and TBK1 with and without rapalog treatment (scale bar = 20 µm). H) Comparison of the Tau flux upon tethering TAX1BP1, ATG13 or TBK1 to Tau aggregates following rapalog treatment. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. I) AF3 prediction of the ternary complex formed by the SKICH domain of TAX1BP1, aa1360-1385 of FIP200 and aa33-75 of NAP1. J) Schematic overview of the ATG13 and TBK1 co-tethering experiment. K) Comparison of the Tau flux upon tethering of ATG13 only or ATG13 and TBK1 co-tethering to the Tau aggregates. Data are presented as mean +/- s.d. (n = 3 biologically independent experiments). Two-way ANOVA with Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. ns, not significant.

Article Snippet: The following primary antibodies and dilution were used in this study: rabbit anti-Tau (1:2500; MA5-42451, Invitrogen), mouse anti-p62 (1:1000; #610832, Clone 3/p62 lck ligand, BD Bioscience), mouse anti-NBR1 (1:1000; H00004077-M01, Clone 6B11, Abnova), rabbit anti-TAX1BP1 (1:1000; #5105, clone D1D5, CST), rabbit anti-FIP200 (1:1000; #12436, clone D10D11, CST), rabbit anti-TBK1 (1:1000; #3013, CST), rabbit anti-TBK1 pS172 (1:1000; #5483, clone D52C2, CST), rabbit anti-ATG13 (1:1000; #13468, clone E1Y9V, CST), rabbit anti-ULK1 (1:1000; #8054, clone D8H5, CST), mouse anti-LC3B (1:1000; #3868S, clone D11, CST), rabbit ATG13 p-S355 (1:1000; #46329, CST), rabbit anti ULK1 p-S317 (1:1000; #37762, CST), rabbit anti-ULK1 p-S757 (1:1000; #14202, CST), rabbit anti-ATG9 (1:1000; #13509, clone D4O9D, CST), rabbit anti-ATG16L1 (1:1000; #8089, clone D6D5, CST), rabbit anti-ATG14 (1:1000; #5504, CST), mouse anti-GAPDH (1:20000; #G8795, clone 71.1, Sigma) The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse (1:10000, #115-035-003, Jackson ImmunoResearch Labs) and HRP-conjugated polyclonal goat anti-rabbit (1:10000; #111-035-003, Jackson ImmunoResearch Labs).

Techniques: Immunofluorescence, Western Blot, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia

doi: 10.1186/s13046-020-01580-4

Figure Lengend Snippet: Autophagy induction by ULK1 inhibitors in AML cells. a After treatment of MV4;11 cells and U937 cells with 2.5 μM of MRT 68921 for 24 h, cell lysates were subjected to western blotting for p-ULK1, p-ATG13, LC3-I/II and p62. α-Tubulin was used as a loading control. All experiments were performed at least three times independently. b TEM-based ultrastructural examination of MV4;11 cells and U937 cells treated with ULK1 inhibitors (MRT 68921; 2.5 μM, SBI-0206965; 5 μM) for 24 h. The arrowheads and arrows indicate autophagosomes and condensed chromatin, respectively. Autophagosomes were counted in at least three different visual fields. Data are the mean ± SD. CTL, control. c MV4;11 and U937 cells were treated with ULK1 inhibitors (MRT 68921; 2.5 μM, SBI-0206965; 5 μM) for 24 h and fixed. Cells were stained for LC3B (green) and observed by confocal microscopy. Representative micrographs demonstrate the characteristic punctuate staining, which indicates autophagosome formation. Nuclei were stained with DAPI (blue). The arrowheads indicate LC3 puncta. The arrows denote condensed nuclei. LC3 puncta in each cell were counted in at least three different visual fields. Data are the mean ± SD. d MV4;11 and U937 cells were treated with ULK1 inhibitors (MRT 68921; MV4;11, 0.5 μM, U937, 2.5 μM, SBI-0206965; 5 μM) in the presence or absence of autophagy inhibitors 3-MA (5 mM). After incubation for 48 h, the apoptotic fraction was measured by flow cytometry based on Annexin-V/PI exclusion. Data are the mean ± SD

Article Snippet: Rabbit polyclonal antibodies against LC3 and p-ATG13 S318 were obtained from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control, Staining, Confocal Microscopy, Incubation, Flow Cytometry

Journal: Journal of Virology

Article Title: Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

doi: 10.1128/JVI.01537-21

Figure Lengend Snippet: Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibodies against Atg5 (2630), p-AKT (4060), AKT (4685), p-mTOR (5536), mTOR (2983), p-S6 (4858), S6 (2317), p-4E-BP1 (2855), 4E-BP1 (9644), p-AMPK (2535), AMPK (5831), p-TSC-2 (3614), TSC-2 (4308), p-Raptor (2083), Raptor (2280), p-Atg13 (26839), Atg13 (13273), ULK1 (8054), p-ULK1 (Ser757) (14202), p-ULK1 (Ser555) (5869), VPS15 (14580), VPS34 (4263), Beclin1 (3495), p-Beclin1 (84966), Atg14 (96752), Atg16L1 (8089), p62 (16177), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Virus, Infection, Western Blot, Membrane, Quantitative RT-PCR, Knockdown, Transfection, Control