Fig 1: The autophagy receptor TAX1BP1 protects against necroptosis by TLR3 or TLR4 ligands.(A, B) Immunoblots of indicated autophagy receptors in total (A) or NP-40 insoluble fractions (B) of BMDM lysates over 6 hr of LPS/zVAD treatment. (C) Immunoblots confirming CRISPR-mediated deletion of indicated autophagy receptor genes in wild-type BMDMs. (D) Cell death assayed by PI staining and live-cell imaging for 12–16 hr following treatment with indicated ligands. Data in (A, B) are representative of three independent experiment; (C, D) are representative of four independent experiments. **p<0.01, ****p<0.0001. Bar graphs depict mean. NTC = non targeting control gRNA.10.7554/eLife.44452.022Figure 5—source data 1.The autophagy receptor TAX1BP1 protects against necroptosis by TLR3 or TLR4 ligands.
Fig 2: Iron-dependent regulation of NCOA4 differs between cell types.A, immunoblot analyses of NCOA4, TAX1BP1, and β-actin in seven human cell lines treated with or without 25 μg/ml FAC for 12 h. The soluble and insoluble fractions in Triton buffer were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. B, immunoblot analyses of NCOA4, FTH1, FTL, β-actin, and H2B in HEK293T cells treated with 20 μM Dfo (D) or 25 μg/ml FAC (F) for 3 h. The cells were lysed with SDS sample buffer or Triton buffer and analyzed by immunoblotting with the indicated antibodies. Dfo, deferoxamine; FAC, ferric ammonium citrate; HEK293T, human embryonic kidney 293T cell line.
Fig 3: p62 is dynamically recruited to damaged lysosomes in HeLa cells(A) Western blot analysis of lysosomal immunoprecipitation following either no treatment (-) or treatment with 1.0 mM LLOMe (+) for 1 h.(B–F) Quantification of lysosomal immunoprecipitation following normalization to a-LAMP1 intensity in either untreated (Unt) or LLOMe-treated HeLa cells. (B) a-FIP200 band intensities (unpaired t test, p < 0.05, n = 3). (C) a-TAX1BP1 band intensities (unpaired t test, p < 0.001, n = 3). (D) a-p62 band intensities (unpaired t test, p < 0.01, n = 3). (E) a-GABARAP band intensities (unpaired t test, p < 0.05, n = 3). (F) a-LC3B band intensities (unpaired t test, p < 0.001, n = 3).(G) Single z-plane representative images of endogenous a-LAMP1 and a-p62 in HeLa cells treated with ethanol (EtOH) as a vehicle control or LLOMe (at 250 or 750 µM) for 2 h. Cell outlines are traced. White boxes indicate inset image region. Arrowheads indicate regions of co-localization. Scale bars (SB): 10 µm; insets: 2 µm.(H) Quantification of the overlapping area from maximum (max) projections between endogenous a-p62 and a-LAMP1 in cells treated with EtOH or different concentrations of LLOMe (one-way ANOVA, p(EtOH/250) < 0.001, p(EtOH/750) < 0.0001, n = 3 experiments. Number of cells analyzed per condition = 54).(I) Quantification of LAMP1 area from max projections for the experiments shown in (H) (one-way ANOVA, p(EtOH/250) = 0.96, p(EtOH/750) = 0.9766, n = 3 experiments. Number of cells analyzed per condition = 54).(J) Representative images of HeLa cells transiently transfected with LAMP1-KillerRed, LAMP2-BFP, and EGFP-p62. Cell outlines are traced. Blue boxes reflect regions irradiated. Arrowheads reflect location of inset images in (K). SBs: 10 µm.(K) Inset images from (J), showing EGFP-p62 recruitment to lysosomes in HeLa cells at 60 min post-laser irradiation. SBs: 2 µm.(L) Quantification of LAMP1-KillerRed intensities within irradiated or Unt regions from the same cells.(M) Quantification of EGFP-p62 intensities within irradiated or Unt regions.(N) Single z-plane representative images of endogenous a-LAMP1, a-p62, and a-GABARAP/L1/L2 in HeLa cells, which were treated with EtOH or 750 µM LLOMe for 2 h. Cell outlines are traced. White boxes indicate inset region. Arrowheads in inset regions indicate co-localization. SBs: 10 am; insets: 2 am.(O) Quantification of the percentage of a-LAMP1 occupied by a-GABARAPs (unpaired t test, p < 0.01, n = 3 experiments. Number of cells analyzed per condition = 29).(P) Quantification of the percentage of a-GABARAPs rings (in single z-plane) co-localizing with a-p62 (% p62+: 94% ± 0.49%). Data are represented as mean ± SEM.(Q) Quantification of a-LAMP1 area per cell (unpaired t test, p = 0.9117, n = 3 experiments. Number of cells analyzed per condition = 29).All error bars reflect mean ± SEM. See also Video S1.
Fig 4: p62 is necessary and sufficient for lysophagy(A) Representative max projections of a-LC3B in pentaKO HeLa cells. Cells were treated with 750 µM LLOMe for 2 h. Cells were transfected with mCherry-LAMTOR2 and either GFP vector, EGFP-p62, EGFP-NBR1, or EGFP-TAX1BP1; endogenous LC3B was visualized by immunostaining. Cell outlines are traced. SBs: 10 µm.(B) Quantification of a-LC3B intensity at LAMTOR2 puncta across different conditions (one-way ANOVA, p(GFP/p62) < 0.05, p(GFP/NBR1) = 0.9042, p(GFP/TAX1) = 0.9417, n = 6 experiments, Number of cells analyzed per condition = 64).(C) Representative max projections of mCherry-LAMTOR2 and a-LC3B in HeLa cells, which were transiently transfected with control siRNA or p62 siRNA (#1) alongside mCherry-LAMTOR2. HeLa cells were treated with 750 µM LLOMe for 2 h. Cell outlines are traced. SBs: 10 µm.(D) Quantification of normalized a-LC3B intensity at LAMTOR2 puncta across different conditions (Mann-Whitney U test, p < 0.0001, n = 40 cells per condition).(E) Quantification of mCherry-LAMTOR2 area across different conditions (unpaired t test, p = 0.6338, n = 3 experiments. Number of cells analyzed per condition = 40).(F) Representative max projections of endogenous galectin-3 flux assay. Cells were fixed immediately following LLOMe treatment or 15 h post-washout of LLOMe. Cells were transiently transfected with one of two ATG5 siRNAs, one of two p62 siRNAs, or a control siRNA. Cell outlines are traced. SBs: 10 µm.(G) Quantification of the galectin-3 flux assay, indicating the fraction of cells with n > 3 galectin-3 puncta per cell (one-way ANOVA, post-LLOMe: p(control/ATG5#1) > 0.9999, p(control/ATG5#2) > 0.9999, p(control/p62#1) > 0.9999, p(control/p62#2) > 0.9999; post-washout: p(control/ATG5#1) < 0.0001, p(control/ATG5#2) < 0.0001, p(control/p62#1) < 0.0001, p(control/p62#2) < 0.0001, p(ATG5 #1/ATG5#2) = 0.9983, p(p62 #1/p62#2) = 0.5105; n = 3 or 4 experiments).All error bars reflect mean ± SEM. See also Figures S1, S2 and S3.
Fig 5: p62 responds to lysosomal damage in human and rat neurons(A) Representative max projections of a-p62 and LAMP1-mNeonGreen in human iPSC-derived neurons (i3Neurons), which were treated with EtOH or 1.0 mM LLOMe for 2 h. Cell outlines are traced. White boxes indicate inset image region. Arrowheads indicate areas of co-localization. Inset images are single z-plane images. SBs: 10 am; insets: 2 am.(B) Quantification of the overlapping area from max projections between a-p62 and LAMP1-mNeonGreen in cells treated with EtOH or with LLOMe (unpaired t test, p < 0.001, n = 4 experiments. Number of cells analyzed per condition = 54).(C) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.5047, n = 4 experiments. Number of cells analyzed per condition = 54).(D) Representative max projections of images comparing a-TAX1BP1 localization with lysosomes in i3Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 am.(E) Quantification of the percentage of LAMP1 area occupied by a-TAX1BP1in cells treated with EtOH or with LLOMe (unpaired t test, p < 0.05, n = 4 experiments. Number of cells analyzed per condition = 45).(F) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.6003, n = 4 experiments. Number of cells analyzed per condition = 45).(G) Representative max projections of images comparing a-NDP52 localization with lysosomes in i3Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 µm.(H) Quantification of the percentage of LAMP1 area occupied by a-NDP52 in cells treated with EtOH or with LLOMe (unpaired t test, p = 0.0708, n = 4 experiments. Number of cells analyzed per condition = 45).(I) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.4643, n = 4 experiments. Number of cells analyzed per condition = 45).(J) Representative images max projections comparing a-OPTN localization with lysosomes in i3Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 µm.(K) Quantification of the percentage of LAMP1 area occupied by a-OPTN in cells treated with EtOH or with LLOMe (unpaired t test, p = 0.2476, n = 4 experiments). Number of cells analyzed per condition = 50).(L) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.8482, n = 4 experiments. Number of cells analyzed per condition = 50).(M) Representative images of rat hippocampal neurons transiently transfected with LAMP1-KillerRed, LAMP2-BFP, and EGFP-p62. Cell outlines are traced. Blue boxes reflect regions irradiated. Arrowheads reflect location of inset images in Figure 1N. SBs: 10 µm.(N) Inset images of LAMP2-BFP and EGFP-p62 from Figure 1M of rat hippocampal neurons following 45-min post-laser activation. SBs: 2 µm.(O) Quantification of LAMP1-KillerRed intensities within irradiated or untreated regions.(P) Quantification of EGFP-p62 intensities within irradiated or untreated regions.All error bars reflect mean ± SEM. See also Video S2.
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