anti stx17  (Proteintech)

Journal: The EMBO Journal

Article Title: MYO5A-mediated stabilization promotes the acquisition of fusion competence in sealed autophagosomes

doi: 10.1038/s44318-025-00686-9

Figure Lengend Snippet: ( A ) WT and MYO5A KO HeLa cells were transfected with GFP-LC3B and co-transfected with HA-ATG12, HA-VPS33A, Flag-SNAP29, or Flag-STX17, as indicated (in magenta). WIPI2 and MYO5A were detected by antibody staining. Scale bar, 10 µm. Zoomed insets highlight colocalization events between stage-specific markers and LC3, as well as between MYO5A and stage-specific markers. Scale bar, 1 µm. The cell boundary is marked in white and the white boxes within the image indicate the insets, which are shown as magnified views in the rightmost and leftmost corners, indicating events of colocalization. ( B ) Quantification of colocalization events between stage-specific markers and LC3B in WT and MYO5A KO cells using the Colocalization plug-in of ImageJ. Data were plotted as mean ± SEM from three independent experiments ( N = 3, n = 60 cells). Statistical significance was assessed by Mann–Whitney Student’s t -test between the WT and MYO5A KO cells for each marker (**** p < 0.0001, *** p = 0.0002 for ATG12, *** p = 0.0008 for SNAP29). ( C ) WT and MYO5A KO cells were transfected with Flag-VAMP8 and stained for LAMP1. Scale bar, 10 µm. Insets show colocalization events between Flag-VAMP8 and LAMP1. Scale bar, 1 µm. The cell boundary is marked in white, and the white boxes within the image indicate the insets, which are shown as magnified views in the rightmost corner, indicating events of colocalization. ( D ) Quantification of colocalization between LAMP1 and Flag-VAMP8 in WT and MYO5A KO cells. Data represent mean ± SEM from three independent experiments ( N = 3, n = 60 cells). Statistical significance was assessed by an unpaired Student’s t -test (ns, non significant). ( E ) Time-lapse images of WT and MYO5A KO HeLa cells transfected with either mCh-LC3B or GFP-LC3B and co-transfected with mCh-DFCP1 or GFP-VPS33A, as indicated. Scale bar, 10 µm. Insets highlight colocalization events between stage-specific markers and LC3. Scale bar, 1 µm. ( F ) Quantification of colocalization between stage-specific markers and LC3B in WT and MYO5A KO cells using the Colocalization plug-in in ImageJ. Data represent mean ± SEM from three independent experiments ( N = 3, n = 40 cells). Statistical significance was determined by unpaired Student’s t -test (*** p = 0.0001, * p = 0.0286). ( G ) WT and MYO5A KO cells were transfected with Flag-VAMP8 and Flag-SNAP29 and either left untreated or subjected to starvation (EBSS, 2 h). Lysates collected 48 h post-transfection were subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with the indicated antibodies. ( H ) Quantification of the proportion of Flag-SNAP29 and STX17 co-precipitated with Flag-VAMP8 from WT and MYO5A KO cells under basal or starvation conditions. Data were plotted as mean ± SEM from three independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis (SNAP29, WT_GM vs. KO_ EBSS; * p = 0.0319, WT_EBSS vs. KO_ GM; * p = 0.0424, WT_EBSS vs. KO_ EBSS; * p = 0.0145; STX17, WT_EBSS vs. KO_ EBSS; * p = 0.0127). ( I ) Schematic representation of the protease protection assay used to evaluate autophagic cargo entrapment within sealed autophagosomes. p62-tagged cargo inside closed autophagosomes is protected from proteinase K (middle panel), unless Triton X-100 is added (right panel), while untreated lysates serve as a control (left panel). Lysates from HeLa cells transfected with Scr or MYO5A siRNA, or treated with DMSO or PBP, were subjected to combinatorial treatments with proteinase K and Triton X-100 as indicated, followed by immunoblotting with anti-p62. .

Article Snippet: FLAG-Stx17 , #45911 (Addgene), deposited by Noboru Mizushima , pAN13.

Techniques: Transfection, Staining, MANN-WHITNEY, Marker, Immunoprecipitation, Western Blot, Control

Journal: The EMBO Journal

Article Title: MYO5A-mediated stabilization promotes the acquisition of fusion competence in sealed autophagosomes

doi: 10.1038/s44318-025-00686-9

Figure Lengend Snippet: ( A ) Endogenous MYO5A was immunoprecipitated from untreated or EBSS-treated cells using anti-MYO5A antibody. The lysates were probed with antibodies against various stage-specific markers, as indicated ( N = 3). ( B ) Proximity ligation assay (PLA) was performed using antibodies against endogenous LC3 and MYO5A. PLA with only the LC3 or only the MYO5A antibody was performed as experimental controls. The assay was carried out under GM, EBSS, and BafA1 conditions. Scale bar, 10 µm. ( C ) The graph represents the number of PLA-positive puncta in different cohorts quantified using the “Analyze particles” plug-in of ImageJ and plotted as mean ± SEM from three independent experiments ( N = 3, n = 100 cells). Statistical significance was assessed by one-way ANOVA with Kruskal–Wallis post hoc test (*** p < 0.001). ( D ) PLA was performed using antibodies against endogenous LC3 and LAMP1. PLA with only LC3 or only LAMP1 antibody was performed as experimental controls (top panel). PLA was performed in WT and MYO5A KO cells, and MYO5A KO cells transfected with FL or CC–GTD constructs of MYO5A. The graph on the right represents the number of PLA-positive puncta in different cohorts quantified using the “Analyze particles” plug-in of ImageJ and plotted as mean ± SEM from three independent experiments ( N = 3, n = 100 cells). Statistical significance was assessed by one-way ANOVA with Kruskal–Wallis post hoc test (**** p < 0.0001, ns non significant). Scale bar: 10 µm. ( E ) WT and MYO5A KO cells (as indicated) were either left untransfected or transfected with GFP-MYO5A or mEmerald-CC–GTD plasmid constructs and co-transfected with HA-VPS33A or Flag-STX17. Zoomed insets highlight colocalization events between VPS33A or STX17 (top) and LC3, as well as expression of MYO5A constructs in the cell (bottom). The cell boundary is marked in white. Scale bar, 5 µm; Inset scale bar, 1 µm. Quantification of colocalization events between VPS33A ( F ) or STX17 ( G ) and LC3 in WT and MYO5A KO cells using the Colocalization plug-in of ImageJ. Data are plotted as mean ± SEM from three independent experiments ( N = 3, n = 60 cells). Statistical significance was assessed by one-way ANOVA with Kruskal–Wallis post hoc test (**** p < 0.0001, *** p < 0.001, ns non significant).

Article Snippet: FLAG-Stx17 , #45911 (Addgene), deposited by Noboru Mizushima , pAN13.

Techniques: Immunoprecipitation, Proximity Ligation Assay, Transfection, Construct, Plasmid Preparation, Expressing

Journal: Autophagy

Article Title: NK cell-derived GZMB (granzyme B) suppresses glioblastoma radioresistance by blocking SDC1-mediated autophagosome maturation

doi: 10.1080/15548627.2025.2601856

Figure Lengend Snippet: GZMB attenuates SDC1-mediated autophagosome maturation. Untreated or sh Sdc1 transfected U251 or T98G cells were incubated in the presence or absence of purified perforin or perforin+GZMB, and then irradiated for a single dose of 4 Gy X-rays. Cells were harvest in 4 h. (A) the protein levels of SQSTM1 and ratio of LC3 II/LC3 I in GBM cells were evaluated by immunoblot assay. Statistical quantitation was analyzed from 3 independent experiments. (B) the autolysosomes of GBM cells in indicated groups were examined with TEM. Autolysosomes were indicated by white arrows. Scale bar: 50 µm (left) or 10 µm (right). (C) cells were immunostained with antibodies to LC3 and LAMP2. Nuclei were stained with DAPI. Scale bar: 10 µm. Colocalized LC3 and LAMP2 was quantified. (D) endogenous SNAP29 and VAMP8 were precipitated by STX17 in co-IP assays in the SDC1-knockdown cells treated with or without GZMB. * p < 0.05; ** p < 0.01; ns, non-statistical significance.

Article Snippet: STX17 , CST , 31,261 , IB (1:1000) IP (1:100).

Techniques: Transfection, Incubation, Purification, Irradiation, Western Blot, Quantitation Assay, Staining, Co-Immunoprecipitation Assay, Knockdown