Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) HEK 293T cells expressing FLAG-STX17 were infected with different strains of Legionella for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A), followed by western blots with antibodies against FLAG, GST, and ubiquitin. Whole-cell lysates were blotted with antibodies against FLAG and vinculin as a loading control. This experiment was repeated three times with similar results. ( B ) HEK 293T cells expressing V5-SNAP29 were infected with different Legionella strains for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A) followed by western blotting with antibodies against V5, GST, and ubiquitin. Whole-cell lysates were probed with antibodies against V5 and vinculin as a loading control. This experiment was repeated three times with similar results. ( C ) Domain architecture of STX17 and SNAP29 showing the PR-ubiquitination sites. ( D ) A549 cells expressing GFP-tagged STX17, STX17TM or the PR-Ub deficient mutant of STX17 were infected with Legionella strains for 2 h before fixation and immunostaining with a Legionella -specific antibody for analysis by confocal microscopy. Control cells were treated with 300 nM Torin-1 for 4 h. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. Scale bar: 5 µm. Scale bar in inset: 2 µm. ( E ) The number of STX17 + bacteria per cell were counted for ~50 cells taken three different experiments. In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. N = 52, 56 cells taken from three experimental replicates. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 7.43E-8. Scale bar: 5 µm. ( F ) The formation of WT and PR-Ub-deficient SNAP29-GFP puncta was monitored in Legionella -infected cells 4 h post-infection. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) SNAP29 puncta were counted in 50-μm 2 regions of interest using FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 50 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, For the graph SNAP29 puncta/cell: *** P = 6.82E-20. For the graph % cells with SNAP29 recruitment to bacteria: *** P = 2.27E-5 (WT, SNAP29WT vs ΔS, SNAP29WT) *** P = 2.56E-6 (WT, SNAP29WT vs WT, SNAP29mut. Scale bar: 5 µm. .

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Expressing, Infection, Mutagenesis, Western Blot, Ubiquitin Proteomics, Control, Immunostaining, Confocal Microscopy, Bacteria, Software, Two Tailed Test

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) (i) HEK 293 T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. FLAG-STX17 was immunoprecipitated using FLAG resin and analyzed by western blot using antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated 2 times with similar results. (ii) HEK 293T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector and immunoprecipitated as shown in ( B ). The samples were then treated with or without pure DupA for 1 h before western blotting with antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated two times with similar results. ( B ) HEK 293T cells were cotransfected with GFP-SNAP29 and HA-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. GFP-SNAP29 was immunoprecipitated with anti-GFP beads, treated with or without pure DupA for 1 h and analyzed by western blot with antibodies against GFP to detect PR-Ub-modified and unmodified SNAP29. The experiment was repeated two times with similar results. ( C ) GST-STX17 and GST-STX17(S195AS202AS209A) were incubated with or without SdeA in the presence of 1 mM NAD + and ubiquitin for 1 h. The samples were analyzed by western blot using antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. ( D ) GST-STX17 and its PR-Ub-deficient mutant (S195AS202AS209A) were modified with or without SdeA, in the presence of 1 mM NAD+ and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A) ( E ) GST-SNAP29 and its PR-Ub-deficient mutant (S61AS63AS70A) were modified with or without SdeA, in the presence of 1 mM NAD + and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A).

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Modification, Incubation, Ubiquitin Proteomics, Mutagenesis

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: Mass spectra of PR-Ub-modified STX17(1–224). S202, S209 were identified as the modified serine residues by high-resolution ETD mass spectrometry.

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Modification, Mass Spectrometry

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) Mass spectrum and deduced sequence map of PR-Ub-modified SNAP29. ( B ) HEK 293 T cells were transfected with GFP-tagged WT STX17, STX17TM, or the STX17 serine mutant (S195AS202AS209A) followed by Legionella infection for 2 h. STX17 was then immunoprecipitated using anti-GFP beads followed by western blotting with antibodies against GFP and ubiquitin. Cell lysates were analyzed by western blot with an antibody against GFP to check the expression levels of GFP-STX17 constructs. The experiment was repeated three times with similar results.

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Sequencing, Modification, Transfection, Mutagenesis, Infection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Construct

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells expressing STX17-GFP were infected with WT Legionella for 2 h in the presence or absence of 100 nM brefeldin A before fixation and staining the intracellular bacteria by DAPI. STX17-positive bacteria was counted in 50 cells per set, taken from three independent experiments. Error bars indicate SEM. Difference between sets was non-significant from p value calculated by two-tailed, type 3 Student’s t test. Scale bar:10 µm. ( B ) A549 cells expressing STX17-GFP were infected with Legionella (WT/ΔS) for 2 h in the presence or absence of 100 nM wortmannin before fixation and immunostaining with antibodies against Legionella . p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.45E-6(WT and ΔS sets without wortmannin), *** P = 2.05E-5 (WT +/-wortmannin), Graph represents n = 50 cells taken from three experiments, error bars indicate SEM. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) Immuno-electron microscopy of HeLa cells transfected with STX17-GFP and infected with WT Legionella-DsRed for 4 h. Ultrathin cryosection immunogold labeled for STX17-GFP by protein A–10-nm gold. Colors are added by Photoshop: Yellow marks a STX-17.GFP-positive ER cisterna closely aligned with the Legionella (Leg) containing vacuole. Green marks the space between the vacuolar membrane and enclosed Legionella. Bar, 200 nm. ( D ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the STX17 and Legionella antibodies to check for the recruitment of STX17 to intracellular bacteria. White arrows mark intracellular bacteria with STX17 recruitment. The data are means ± SEM of 118 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.21E-6. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( E ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the SNAP29 and Legionella antibodies to check for the recruitment of SNAP29 to intracellular bacteria. White arrows mark intracellular bacteria with SNAP29 recruitment. The data are means ± SEM of 120 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 2.21E-4. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( F ) HeLa cells were cotransfected with RFP-tagged SdeA or its catalytic mutant (E860AE862A) and GFP-tagged WT SNAP29 or its PR-Ub-deficient mutant. Cells were treated with 300 nM Torin-1 for 4 h to induce autophagy before fixation and confocal imaging. Scale bar:5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) The graph shows the number of cells with SNAP29-GFP puncta (from panel d) counted in FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 30 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 0.00032. In bar graph, the data are means ± SEM of n > 30 cells from three independent experiments. Scale bar:5 µm. ( H ) A549 cells were treated with SNAP29 or control siRNA for 48 h followed by infection Legionella. Intracellular bacterial replication was assessed after 0, 24 and 48 h. Data are means ± SEM of three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 2.1E-4 (ΔR), ** P = 0,.031(ΔRΔS). (ni not infected, WT wild-type, Legionella , ΔS-ΔSidE Legionella ).

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Expressing, Infection, Staining, Bacteria, Two Tailed Test, Immunostaining, Immuno-Electron Microscopy, Transfection, Labeling, Membrane, Mutagenesis, Imaging, Software, Control

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells were infected with WT or ΔS Legionella for 1 h, fixed and immunostained with the indicated antibodies to check for the recruitment of endocytic and autophagic markers to intracellular bacteria. Scale bar: 5 µm. White arrows mark intracellular bacteria. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( B ) The experiment in ( A ) was quantified to measure the recruitment of each protein to intracellular bacteria. The total number of intracellular bacteria per cell and the number of bacteria which is surrounded by the protein marker were counted manually in FIJI to find the % of intracellular bacteria which were positive for the recruitment of the protein. The data are means ± SEM of 50 cells representing three experiments. P value was calculated using a two-tailed type 3 Student's t test. *** P = 3.15E-5 (STX17), ** P = 0.0066 (ULK2), ** P = 0.01 (ATG14L), ** P = 0.001(FIP200), n.s. P = 0.42(Rab5). Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) Proximity labeling of bacterial vacuoles in digitonin-permeabilized cells 2 h post-infection. ( D ) Western blots of the indicated proteins after streptavidin pulldown from lysates derived from cells treated with TurboID-ProtA and Legionella antibody. The data represent means ± SD taken from three independent experiments. P value was calculated using a two-tailed type 3 Students t test. n.s. P = 0.44 (Rab5), n.s. P = 0.802 (EEA1), * P = 0.013 (STX17), ** P = 0.006 (FIP200), * P = 0.03(ATG14L), n.s. P = 0.387 (ATG5), n.s. P = 0.523 (ATG12), n.s. P = 0.714 (CANX). ( E ) HeLa cells expressing CD32 (for efficient uptake of Legionella) were uninfected (n.i.) infected with WT or ΔS Legionella for 6 h, fixed and prepared by following a protocol which was similar to that in ( C ), and imaged by TEM. In total, 30 images collected from three experiments were analyzed to count number of large electron-dense bacterial vacuoles and the number of early phagosome-like vacuoles. Error bars represent ± SD. P value was calculated using two-tailed type 3 Student’s t test, *** P = 1.008E-8. Arrows mark intracellular bacteria in lysosome-like vesicles (in WT) or in smaller phagosomes (in ΔS) (WT wild-type, Legionella , ΔS-ΔSidE Legionella ). .

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Infection, Bacteria, Marker, Two Tailed Test, Labeling, Western Blot, Derivative Assay, Expressing

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) HeLa cells expressing CD32 (for efficient uptake of Legionella ) were infected with WT Legionella for 2 h or 6 h. Cells were fixed using 2.5% glutaraldehyde in 0.1 M cacodylate buffer for two hours at RT. Cells were scraped of the petridish, post-fixed with 1% reduced osmium tetroxide at RT, dehydrated and embedded using EPON. Ultrathin sections (50 nm) were imaged by transmission electron microscopy. Arrows mark intracellular bacteria, Lyl-lysosome-like organelles. ( B ) HeLa cells expressing APEX2-FLAG-STX17 are infected with WT Legionella for 2 h followed by fixing cells and staining cells with FLAG antibody to check its recruitment to intracellular bacteria. DAPI marks nuclear DNA and cytosolic bacteria. Scale bar:5 µm. White arrows indicate intracellular bacteria. ( C ) Lysates used as input in streptavidin IP shown in Fig. . ( D ) Volcano plot showing how the biotin-labeled proteome changes when HeLa cells expressing APEX-STX17 are infected with WT Legionella for 2 h; GO analysis of the biotin-labeled proteome showing pathways upregulated by infection with WT Legionella . Red and green indicate compartments containing proteins enriched following infection with WT Legionella and in uninfected cells, respectively. Data represent mean fold change of three experimental replicates per infection set ( n = 3). P value was calculated using two-tailed type 3 Student’s t test and significant candidates were chosen having P value ≤ 0.01 and log2(fold change) value minimum of ±0.5. ( E ) Volcano plot showing changes in the biotin-labeled proteome following the infection of HeLa cells expressing APEX-STX17 with WT and ΔS Legionella for 2 h. GO analysis of the biotin-labeled proteome showing pathways upregulated by infection with WT vs ΔS Legionella . Data represents mean fold change of three experimental replicates per infection set ( n = 3). P value was calculated using two-tailed type 3 Student’s t test and significant candidates were chosen having P value ≤ 0.01 and log2 (fold change) value minimum of ±0.5. Red and green indicate compartments containing proteins enriched following infection with WT and ΔS Legionella , respectively. (ni not infected, WT wild-type Legionella , ΔS-ΔSidE).

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Expressing, Infection, Transmission Assay, Electron Microscopy, Bacteria, Staining, Labeling, Two Tailed Test

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells were treated with control or STX17 siRNA for 48 h followed by infection with WT Legionella . Cells were fixed and stained for the indicated autophagy markers 1 h after infection. The data are means ± SEM of 50 cells representing three experiments. which were analyzed per sample to quantify recruitment of FIP200 and ATG14L to bacteria. P value was calculated using two-tailed type 3 Student’s t test, *** P = 5.83E-7 (FIP200), *** P = 1.92E-12 (ATG14L), Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( B ) A549 cells were treated with STX17 or control siRNA for 48 h followed by infection with WT or ΔS Legionella for 12 h (MOI = 1). Cells were fixed for immunostaining with a Legionella -specific antibody followed by confocal microscopy. The LCV size was estimated in FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. n = 32, 31 cells taken from three independent experiments. p value was calculated using two-tailed, type 3 Student’s t test, *** P = 8.13E-16. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) A549 cells were treated with control or STX17 siRNA for 48 h followed by infection with WT or ΔS Legionella . Intracellular bacterial replication was assessed after 0, 24, and 48 h. Data are means ± SEM of three independent experiments. P value was calculated using two-tailed type 3 Student’s t test, ** P = 0.00526 (WT, control vs STX17siRNA, 24 h), ** P = 0.00815 (WT, control vs STX17siRNA, 48 h). ( D ) A549 cells were treated with STX17 or control siRNA for 48 h followed by transfection with WT or PR-Ub-deficient STX17 for 24 h. Intracellular bacterial replication was assessed after 0, 24, and 48 h. Data are means ± SEM of three independent experiments ** P = 0.0077 (WT, STX17siRNA versus STX17 mutant, STX17siRNA, 48 h). Western blotting with STX17 antibody shows knockdown efficiency of STX17 siRNA and reconstitution with WT or mutant STX17. ( E ) Proximity labeling assay workflow. HeLa cells expressing doxycycline-inducible APEX-STX17 and CD32(for increasing the efficiency of Legionella uptake) were infected with Legionella for 2 h before treatment with biotin-tyramide and H 2 O 2 followed by streptavidin pulldown. The samples were reduced, alkylated and digested with trypsin before MS analysis. Samples representing three biological replicates each of non-infected and Legionella -infected cells were analyzed in a single reaction by 6-plex TMT labeling. ( F ) Volcano plot showing changes in the biotin-labeled proteome following the infection of HeLa cells expressing APEX2-FLAG-STX17 with ΔR and ΔRΔS Legionella for 2 h, GO analysis of the biotin-labeled proteome showing pathways upregulated by infection with ΔR vs ΔRΔS Legionella . Data represents mean fold change of three experimental replicates per infection set ( n = 3). P value was calculated using two-tailed type 3 Student’s t test and significant candidates were chosen having P value ≤ 0.01 and log2 (fold change) value minimum of ±0.5. Red and green indicate compartments containing proteins enriched following infection with ΔR and ΔRΔS Legionella , respectively. ( G ) Cells expressing doxycycline-inducible APEX-STX17 were infected with Legionella for 2 h before treatment with biotin-tyramide and H 2 O 2 followed by streptavidin pulldown. The samples were analyzed by western blot with antibodies against proteins of the autophagic and endosomal pathways. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. WT vs ΔS P values: * P = 0.01006 (FIP200), ** P = 0.0206 (ULK1), P = ***0.0007 (ATG13) ** P = 0.005 (ATG14), * P = 0.0111(Beclin1), * P = 0.0219 (WIPI2), ** P = 0.0038 (ATG5), * P = 0.018 (ATG12), ** P = 0.001 (ATG16), ** P = 0.0036 (VAMP8), * P = 0.0424 (SNAP29). (n.i. not infected, WT wild-type, Legionella , ΔS ΔSidE Legionella ). .

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Control, Infection, Staining, Bacteria, Two Tailed Test, Immunostaining, Confocal Microscopy, Software, Transfection, Mutagenesis, Western Blot, Knockdown, Labeling, Expressing

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) PR-Ub (PDB ID: 5M93) was manually attached to residue S63 of SNAP29 in the structure of the STX17-SNAP29-VAMP8 complex (7BV6) using Pymol. ( B ) Schematic showing the formation of the autophagosomal SNARE complex, and the effect of PR-ub on its formation. ( C ) Untagged SNAP29 and STX17-GST was purified from E. coli and incubated with His-tagged Ub in an in vitro PR-ub assay. PR-Ub modified STX17 and SNAP29 were enriched by nickel-NTA-based affinity purification and incubated with 100 µg protein lysate from HEK 293T cells. This was followed by GST-pulldown of STX17 and antibody-based immunoprecipitation of SNAP29 to check for alterations in protein-protein interactions upon PR-Ub. The data represents means ± SD of three independent experiments P value was calculated using two-tailed type 3 Students t test 0.001 < ** P ≤ 0.01). ** P = 0.004 (VAMP8), ** P = 0.001 (ATG14L), ** P = 0.022 (PR-Ub STX17). ( D ) WT and PR-Ub-modified SNAP29 were incubated with equimolar amounts of purified GST-STX17, VAMP8 and ATG14L. Interactors of PR-Ub-modified SNAP29 were observed by immunoblotting. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.0068 (STX17), ** P = 0.0059 (VAMP8). ( E ) WT or PR-Ub modified GST-STX17 were incubated with equimolar amounts of purified GST-STX17, VAMP8 and ATG14L. Interactors of PR-Ub modified STX17 were observed by immunoblotting. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.042 (ATG14L), ** P = 0.029 (SNAP29). ( F ) WT or PR-Ub modified forms of both STX17 and SNAP29 were incubated with VAMP8 and ATG14L followed by immunoprecipitation of GST-STX17 and immunoblotting to check for protein-protein interactions. Pure proteins were run as the reaction inputs and immunoblotted with indicated antibodies. The data represent means ± SD of three independent experiments. P value was calculated using two-tailed type 3 Students t test. ** P = 0.0208 (ATG14L), ** P = 0.023 (VAMP8), *** P = 0.00046 (SNAP29). ( G ) Schematic showing the effect of PR-Ub of STX17 and SNAP29 on bacterial vacuole formation. PR-Ub of STX17 enhances interactions with ATG14L which results in the recruitment of ER membranes to the bacterial vacuole.PR-Ub of SNAP29 prevents fusion of the STX17-positive vacuoles with the VAMP8-positive lysosomes. .

Article Snippet: STX17-ser mutant , Lab generated using STX17 coding sequence of STX17 from addgene #45911 , .

Techniques: Residue, Purification, Incubation, In Vitro, Modification, Affinity Purification, Immunoprecipitation, Protein-Protein interactions, Two Tailed Test, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-7 inhibits progression of glioblastoma by impairing autophagy resolution, energy metabolism and ECM remodeling

doi: 10.1186/s13046-025-03504-6

Figure Lengend Snippet: MiR-7 posttranscriptionally regulates the expression of the SNARE complex proteins STX17 and SNAP29. A . qRT-PCR analysis of hSTX17 and hSNAP29 mRNA expression levels in U87-MG cells overexpressing control mimic (CM) or miR-7-5p (miR-7). B . Representative Western blots of hSTX17 and hSNAP2 in cells treated as in A. HSP90 was used as a loading control. Right graph shows relative protein expression normalized to HSP90. Data correspond to the means ± SEM of three experiments performed in triplicate. *** P < 0.001, significantly different from cells transfected with CM (normalized to 1). C . Luciferase reporter activity of STX17 3’UTR in HEK293 cells transfected with CM or miR-7 and with 3’UTRs with wild-type (WT) or the constructs containing the indicated point mutations (PM) or double point mutations (DPM) (upper panels, highlighted in red). Data are expressed as relative luciferase activity compared with the activity in control samples cotransfected with an equal concentration of CM and correspond to the means ± SEM of three experiments performed in triplicate. ** P < 0.01, significantly different from cells cotransfected with CM and the WT or PM or DPM vectors of the 3′UTRs. D . Luciferase reporter activity of SNAP29 3’UTR as indicated in C. * P < 0.05, *** P < 0.001. E-F . Rescue of autophagy flux blockade induced by miR-7. Representative Western blot of autophagy markers p62 and LC3BI/II in cell transfected with CM or miR-7 and a control GFP plasmid or a plasmid expressing STX17 ( E , STX17-GFP) and SNAP29 ( F , SNAP29-GFP) ORF regions lacking their 3’UTR. Cells were treated with 200nM of CQ for 6 h. HSP90 was used as loading control. ( E , F , right panels). Immunofluorescence confocal imaging cells transfected the STX17-GFP ( E ) and SNAP29-GFP ( F ) plasmids showing colocalization with endogenous STX17 or SNAP29 (red). Dapi (blue) was used to stain the nucleus. Scale bar: 10 μm

Article Snippet: 20–30 μg of protein were analyzed by SDS-PAGE and transferred onto nitrocellulose membranes that were blocked with 5% BSA (w/v) in PBS and probed with the following antibodies from Cell Signaling [LC3B (2775), ULK1 (8054T), AKT (9272), p-AKT (9271), mTOR (2972), p-mTOR (2971), S6K (9202), p-S6K (9205), AMPK (2532), p-AMPK (2535) and CTSB (31718T), ENO2 (24330), LGALS8 (45586), PDK1 (3062)], from Abcam [p62 (ab56416), LAMP1 (ab208943), LAMP2 (ab25631) and VDAC1 (ab186321)], STX17 (NBP1-93968) from Novus, as well as RAB7 (376362) and EGFR (53274) from Santa Cruz Biotechnology.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Luciferase, Activity Assay, Construct, Concentration Assay, Plasmid Preparation, Immunofluorescence, Imaging, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-7 inhibits progression of glioblastoma by impairing autophagy resolution, energy metabolism and ECM remodeling

doi: 10.1186/s13046-025-03504-6

Figure Lengend Snippet: Squematic summary of miR-7 effects on GBM. A . Schematic representation of the proposed mechanism of action of miR-7 on autophagy. Red arrows show the steps where miR-7 has its influence or main effect. A detail of the STX17–SNAP29–VAMP8 SNARE complex is shown as well to further illustrate the role of SNAP29 and STX17 in the autophagosome and lysosome fusion. B. Schematic representation summarizing the effects miR-7 on GBM tumors, ECM remodeling and energetic metabolism. Red arrows indicate up- or down-regulation of specific functions exerted by miR-7. llustration were created with BioRender ( https://biorender.com )

Article Snippet: 20–30 μg of protein were analyzed by SDS-PAGE and transferred onto nitrocellulose membranes that were blocked with 5% BSA (w/v) in PBS and probed with the following antibodies from Cell Signaling [LC3B (2775), ULK1 (8054T), AKT (9272), p-AKT (9271), mTOR (2972), p-mTOR (2971), S6K (9202), p-S6K (9205), AMPK (2532), p-AMPK (2535) and CTSB (31718T), ENO2 (24330), LGALS8 (45586), PDK1 (3062)], from Abcam [p62 (ab56416), LAMP1 (ab208943), LAMP2 (ab25631) and VDAC1 (ab186321)], STX17 (NBP1-93968) from Novus, as well as RAB7 (376362) and EGFR (53274) from Santa Cruz Biotechnology.

Techniques:

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 2. p62 FIR Phosphorylation Enhances FIP200 Binding (A) Overview of phosphosites in the FIR of affinity-purified p62 as identified by mass spectrometry (Figures S2A and S2B). (B) Phospho-mimicking mutations were introduced in GST-p62 FIR as follows: 1P (S349D); 3P (S365D, S366D, S370D); 4P (S349D, S365D, S366D, S370D). GSH beads were coated with GST or the GST-p62 FIRs, incubated with GFP-FIP200 CTR (aa 1458–1594) and imaged by microscopy at equilibrium. GFP signals on the beads were normalized to the signal of GFP-FIP200 CTR bound to GST-p62 FIR 4P. Average intensity and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. The same beads were analyzed by SDS-PAGE like in a pull-down experiment (Figure S2D). (C) The binding of GFP-LC3B to GST-p62 phospho-mimicking mutants was assessed like in (B). (D) GSH beads were coated with full-length GST-FIP200 and incubated with full-length mCherry-p62 WT or the 4P phospho-mimicking mutant. Beads were imaged by microscopy. The mCherry signal is shown in false color (ImageJ: Fire).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Phospho-proteomics, Binding Assay, Mass Spectrometry, Incubation, Microscopy, SDS Page, Mutagenesis

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 3. LC3B Competes with FIP200 for p62 Binding, and FIP200 Is Excluded from the Autophagosomal Lumen (A) RFP trap beads were coated with mCherry-p62 FIR 4P and incubated with the GFP-FIP200 CTR (aa 1458-1594, 1.1 mM). Then, increasing concentrations of LC3B were added. Beads were imaged by microscopy. The intensities of the GFP and mCherry signals on the beads were measured and plotted against the LC3B concentrations (lower panels). A zoom-in of the plot at the lowest LC3B concentrations is shown on the left. Average intensities and SD for n = 3 are shown. Negative and positive controls of binding are shown in Figure S3B. (B) HeLa cells were left untreated, starved or treated with puromycin both in presence or absence of bafilomycin. Cell lysates were then treated with proteinase K in the presence or absence of Triton X-100 and analyzed by western blotting with anti-p62 and anti-STX17. LC3B processing was used to monitor autophagy induction. The percentage of protease protection for FIP200, in comparison to p62 and STX17 is plotted on the right. Average protection and SD for n = 3 are shown. Protease protection in starved cells treated with wortmannin and/or bafilomycin are shown in Figure S3D. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Binding Assay, Incubation, Microscopy, Western Blot, Comparison

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 4. Crystal Structure of FIP200 CTR (A) Crystal structure of the FIP200 CTR monomer. The molecule comprises a helix, a linker, and a globular Claw domain. The structure is colored according to its secondary structure elements (helix, purple; b strands, orange; loops, gray). (B) Close-up of the Claw domain. The fold resembles a Claw with the b sheet being the palm of the Claw and loops L2, L4, and L5 flexed fingers. Same view and coloring used in (A). (C) Topology plot of monomeric FIP200 CTR. a helices are shown as purple cylinders and b strands as orange arrows. (D) Dimerization of FIP200 CTR. The homodimer is formed via two interfaces: interface 1 (blue box) and interface 2 (red box). Monomers are colored in orange and green. Interface residues are shown as sticks and labeled for only one monomer. (E) Surface conservation plot of the FIP200 CTR monomer based on the sequence alignment of 11 different species (Figure S5B). Conserved residues are colored in purple and non-conserved residues in cyan. The second monomer is shown in cartoon representation. (F) Analytical size-exclusion chromatography coupled to right-angle light scattering. Absorption at 280 nm (blue) and oligomeric state (as apparent molecular weight, divided by molecular weight of the monomer) (red) are plotted against the retention volume (mL). See also Figures S4 and S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Labeling, Sequencing, Size-exclusion Chromatography, Molecular Weight

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 5. p62 LIR Motif Binding Depends on a Positively Charged Pocket in FIP200 CTR (A) Electrostatic surface potential of the FIP200 Claw domain. Positively and negatively charged surfaces are colored in blue and red, respectively. The coor- dination of sulfate ions and amino acids of interest are shown as sticks. (B) GSH beads were coated with GST-p62 FIR 4P, incubated with the indicated GFP-FIP200 CTR (aa 1458–1594) mutants and imaged by microscopy. For each sample the GFP intensity was normalized to the signal of GFP-FIP200 CTR WT on GST-p62 FIR 4P-coated beads. Average intensity and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. Protein inputs are shown in Figure S5C. (C) mCherry-p62 (2 mM) was incubated with GST-4x ubiquitin (10 mM) to form condensates in solution. Pre-formed condensates were incubated with 1 mM GFP- FIP200 CTR (aa 1458–1594). The recruitment of GFP-FIP200 CTR to p62-ubiquitin clusters was monitored by confocal microscopy. Scale bar, 5 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Binding Assay, Incubation, Microscopy, Ubiquitin Proteomics, Confocal Microscopy

Journal: Molecular cell

Article Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

doi: 10.1016/j.molcel.2019.01.035

Figure Lengend Snippet: Figure 6. p62-FIP200 Interaction in Cells (A) Colocalization analysis of p62 and FIP200 in HAP1 cells (WT or ATG7KO) left untreated or treated with wortmannin (1 mM) for 1 h. Endogenous p62 and FIP200 were detected by immunofluorescence. Scale bar, 10 mm. Average percentages of colocalization and SEM for n = 3 are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. (B) Immunofluorescence of p62 and FIP200 in HAP1 STG-p62 cell line left untreated or treated with bafilomycin (400 nM) for 1 h. p62 was detected through the GFP tag fused to the endogenous protein and FIP200 was detected by immunofluorescence. Scale bar, 10 mm. Average percentages of colocalization and SEM (n = 2) are shown. Significant differences are indicated with * when p value % 0.05, ** when p value % 0.01, and *** when p value % 0.001. (C) The C terminus of endogenous FIP200 was tagged in HAP1 cells with GFP-TEV-Strep (FIP200-STG). Affinity purification was performed using HAP1 WT or FIP200-STG cells and the bound material was analyzed by western blotting with anti-p62. The intensities of the p62 bands were normalized for the total level of p62 in the lysate (input). Average p62 levels and SD for n = 4 are shown. Three additional replicates of the immunoprecipitation are shown in Figure S6C.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-p62 BD Bioscience Cat#610832; RRID: AB_398152 Mouse anti-GST Sigma Cat#SAB4200237; RRID: AB259845 Rabbit anti-FIP200 (D10D11) Cell Signaling Technology Cat#12436 Rabbit anti-FIP200 Atlas antibodies Cat#HPA053049; RRID: AB_2682025 Mouse anti-FIP200 (6B2) MFPL antibody facility N/A Rabbit anti-Stx17 Sigma Cat#HPA001204; RRID: AB_1080118 Mouse anti-LC3B (clone 2G6) nanoTools Cat#0260-100 Rabbit anti-GABARAP Cell Signaling Technology Cat#13733 Mouse anti-GFP Roche Cat#11814460001; RRID: AB_390913 Mouse anti-myc (clone 4A6) Millipore Cat#05-724; RRID: AB_310809 Rabbit anti-Atg19 Eurogentec N/A Rabbit anti-Ape1 Gift from Kraft lab (Institute for Biochem.

Techniques: Western Blot, Immunoprecipitation

Journal: Thoracic Cancer

Article Title: SLC34A2 promotes cell proliferation by activating STX17 ‐mediated autophagy in esophageal squamous cell carcinoma

doi: 10.1111/1759-7714.15314

Figure Lengend Snippet: Solute carrier family 34 member 2 (SLC34A2) promotes esophageal squamous cell carcinoma (ESCC) cell autophagy and proliferation by upregulating STX17. (a) Immunoprecipitation (IP) combined with Coomassie brilliant blue staining and mass spectrometry was used to identify the interacting proteins of SLC34A2. (b) The interaction between three top‐ranking autophagy‐related proteins and SLC34A2 was tested by IP and western blot (WB) in 293 T cells transfected with SLC34A2‐HA or vector‐HA. (c) Co‐IP was used to verify the SLC34A2‐STX17 interaction in ESCC cells. (d) The STX17 protein level was examined in SLC34A2‐ overexpressed/−silenced cells by WB. (e) WB was utilized to examine the levels of LC3B and P62 in SLC34A2‐overexpressed ESCC cells under starvation conditions after silencing STX17. (f) The mRFP‐GFPLC3B reporter was performed to monitor the autophagic flux of SLC34A2‐overexpressed ESCC cells with transient STX17 knockdown. Scale bar, 5 μm. (g, h) Cell counting kit‐8 (CCK‐8) and 5‐ethynyl ‐2′‐deoxyuridine (EdU) assays were used to detect the proliferation ability of SLC34A2‐overexpressed cells with transient STX17 knockdown. Scale bar, 20 μm. (i) Five nude mice in each group were subcutaneously injected with KYSE520‐SLC34A2 cells with STX17 knockdown, KYSE520‐SLC34A2 cells and KYSE520 wild‐type cells. The mean tumor volumes (cm 3 ) and weights (g) were measured 3 weeks later. The grayscale value of the target protein in the WB was quantified using ImageJ software and normalized to α‐tubulin. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The siRNAs targeting STX17 (NCBI Gene: 55014; siSTX17‐2#, 5′‐GGGACAAGTTGCATGAAGA‐3′; siSTX17‐3#, 5′‐CCGAAAGGATGACCTAGTA‐3′) and a negative control were designed and constructed by RiboBio (China).

Techniques: Immunoprecipitation, Staining, Mass Spectrometry, Western Blot, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Knockdown, Cell Counting, CCK-8 Assay, Injection, Software

Journal: Thoracic Cancer

Article Title: SLC34A2 promotes cell proliferation by activating STX17 ‐mediated autophagy in esophageal squamous cell carcinoma

doi: 10.1111/1759-7714.15314

Figure Lengend Snippet: Solute carrier family 34 member 2 (SLC34A2) inhibits the ubiquitination and degradation of STX17 by interacting with STX17. (a, b) SLC34A2‐overexpressed/−silenced cells were treated with translational inhibitor cycloheximide (CHX, 100 μmol/L) at various time points before being collected for western blot (WB). The expression levels of STX17 were normalized against loading control α‐tubulin. (c, d) The stability of STX17 protein was evaluated by the ubiquitination assay in SLC34A2‐overexpressed/−silenced cells after treatment with MG132. *** p < 0.001.

Article Snippet: The siRNAs targeting STX17 (NCBI Gene: 55014; siSTX17‐2#, 5′‐GGGACAAGTTGCATGAAGA‐3′; siSTX17‐3#, 5′‐CCGAAAGGATGACCTAGTA‐3′) and a negative control were designed and constructed by RiboBio (China).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Control

Journal: Nature Communications

Article Title: Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17

doi: 10.1038/s41467-025-55906-x

Figure Lengend Snippet: a , b Co-IP analysis of the endogenous HOPS subunits, STX17, VAMP7 and VAMP8 with HA-SNAP47 (WT, 3KR or 3KQ) in SNAP47 KO HeLa/Parkin cells ( a ). This result is part of Supplementary Fig . The proteins levels were quantified by ImageJ software and normalized with HA-SNAP47 ( b ). c , d HeLa cells stably expressing GFP-SNAP47 (WT, 3KR or 3KQ) were cultured in EBSS for 2 h, then stained with antibody against LC3 ( c ). Scale bar, 5 μm. The percentage of GFP-SNAP47-positive LC3 puncta in total LC3 puncta is shown ( d ). n = 3, 100 cells from three independent experiments. e , f HeLa/Parkin cells stably expressing mito-BFP and GFP-SNAP47 (WT, 3KR or 3KQ) were treated with OA for 4 h, then stained with with antibody against LC3 ( e ). Scale bar, 5 μm. The percentage of GFP-SNAP47 + LC3 + mito-BFP + puncta in total LC3 + mito-BFP + puncta is shown ( f ). n = 3, 100 cells from three independent experiments. g Schematic for the experimental procedures used in the reconstituted fusion reactions. h , i The effect of SNAP47 acetylation on fusion activity between proteoliposomes reconstituted with STX17 and VAMP8 ( h ). neg.ctrl. denotes no addition of SNAP29 and SNAP47. Statistics of the liposome fusion in ( h ) at 2400 s was shown ( i ). j , k The in vitro interaction of purified GST-SNAP47 (WT or mutants) with His-STX17 and VAMP8 ( j ) or His-STX17 and His-VAMP7 ( k ). l Schematic for the experimental procedures used in the SNARE complex assembly assay. m-o Assembly of the SNARE complex containing STX17, SNAP47 (WT, 3KR or 3KQ), and VAMP7/VAMP8 analyzed by FRET assays ( m , n ). Statistics of EPR (relative proximity ratio of FRET) at 2,500 s were shown ( o ). Data in ( b – o ) are presented as mean ± SD, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA ( b , o ) or one-way ANOVA ( d , f ). P values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Purified STX17 SNARE domain (residues 161−231, E198C), VAMP7 SNARE domain (residues 123−187, E152C), and VAMP8 SNARE domain (10−74, E39C) were conjugated with 3-fold molar ratio of ATTO647 maleimide (ATTO-TEC GmbH, #AD 550-45) and ATTO550 maleimide (ATTO-TEC GmbH, #AD 647N-45), respectively, according to the manufacturer’s instruction.

Techniques: Co-Immunoprecipitation Assay, Software, Stable Transfection, Expressing, Cell Culture, Staining, Activity Assay, In Vitro, Purification

Journal: Nature Communications

Article Title: Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17

doi: 10.1038/s41467-025-55906-x

Figure Lengend Snippet: a Co-IP analysis of interactions between the Flag-tagged HOPS subunits with HA-SNAP47 or HA-SNAP29 in HEK293T cells. PI line is for indicating the position of HA-SNAP47 and HA-SNAP29. n = 3 independent experiments. b Co-IP analysis of interactions between the endogenous HOPS subunits with Flag-SNAP47 or Flag-SNAP29 in HEK293T cells. n = 3 independent experiments. c In vitro interactions of purified GST-SNAP47 with His-tagged HOPS subunits. n = 3 independent experiments. d The central ionic layer of SNAP47 was identified through sequence alignment, participating residues glutamine-143 and glutamine-390 in SNAP47 are indicated by red color. The PH domain, SNARE motif, and second SNARE motif are colored by blue, green, and laurel-green, respectively. e, f Co-IP analysis of the endogenous HOPS subunits, STX17, VAMP7 and VAMP8 with HA-SNAP47 (WT or 2QR-Q143R/Q390R) in SNAP47 KO HeLa/Parkin cells ( e ). This result is part of Supplementary Fig . The proteins levels were quantified by ImageJ software and normalized with HA-SNAP47 ( f ). g – j WT and SNAP47 KO HeLa/Parkin cells were treated with OA for 4 h. Following separation via OptiPrep membrane flotation, autophagosome-enriched fractions (light-density fractions 3–5) were analyzed by immunoblotting using the indicated antibodies ( g ). The proteins levels were quantified by ImageJ software and normalized with LC3 ( h – j ). k – n WT, SNAP47 KO, and SNAP47 WT or Flag-SNAP47 LIR mutant complemented SNAP47 KO HeLa/Parkin cells were treated with OA for 4 h. Following separation via the OptiPrep membrane flotation, autophagosome-enriched fractions (light-density fractions 3–5) were analyzed by immunoblotting using the indicated antibodies ( k ). The proteins levels were quantified by ImageJ software and normalized with LC3 ( l – n ). Data in ( f – n ) are presented as mean ± SD, n = 3 independent experiments, statistical significance was assessed by a two-way ANOVA. P values are indicated in the figure. Source data are provided as a Source Data file.

Article Snippet: Purified STX17 SNARE domain (residues 161−231, E198C), VAMP7 SNARE domain (residues 123−187, E152C), and VAMP8 SNARE domain (10−74, E39C) were conjugated with 3-fold molar ratio of ATTO647 maleimide (ATTO-TEC GmbH, #AD 550-45) and ATTO550 maleimide (ATTO-TEC GmbH, #AD 647N-45), respectively, according to the manufacturer’s instruction.

Techniques: Co-Immunoprecipitation Assay, In Vitro, Purification, Sequencing, Software, Membrane, Western Blot, Mutagenesis