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: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

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: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

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: ( 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: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

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: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

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

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: Mapping the cellular localization of BDCPs. (A) HeLa cells stably transfected with LYST tagged with a tandem dimer NeonGreen were treated for 30 min with 20 nM of Lysotracker Deep Red and imaged live (upper panel) or fixed and stained with an anti-EEA1 antibody (lower panel). Scale bar 10 µm (left merged) or 1 µm (right merged). (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL2 and mScarlet-I-RAB25 were imaged live. Scale bar 10 µm (left merged) or 1 µm (right merged). (C) HeLa cells stably transfected with 3xFlag-EGFP-ALFY and mScarlet-I-RAB8A were imaged live. Scale bar 10 µm (left merged) or 1 µm (right merged). (D) HeLa cells stably transfected with 3xFlag-EGFP-ALFY and mScarlet-RAB6A were imaged live. ALFY speckle co-migrates with the tip of RAB6A-positive tubule (arrows). Scale bar 10 µm (main figure) or 1 µm (inset magnifications). (E) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet tagged RAB4A, RAB6A, RAB11A, or RAB14 were imaged live. NBEA partially colocalizes with the tubular segments of RAB4A-, RAB11A,- or RAB14- positive endosomes and RAB6A-positive Golgi-connected tubules (arrows). Scale bar 10 µm (left merged) or 1 µm (right merged). (F) HeLa cells stably transfected with the indicated small GTPases were fixed and stained with an anti-LRBA antibody. Endogenous LRBA colocalizes with mScarlet-RAB6A, mScarlet-RAB11A and ARF1-mScarlet on cytosolic tubules (arrows). Scale bar 10 µm (left merged) or 1 µm (right merged). (G) HeLa cells fixed 3 min after the addition of 10 µg/ml of AlexaFluor647-Transferrin to the culture media and stained with an anti-LRBA antibody. Scale bar 10 µm (upper merged) or 1 µm (middle and lower merged. (H) Examples of mScarlet-RAB6A-positive Golgi and Golgi-connected tubules that colocalize with endogenous LRBA as in F upper panel. Scale bars 1 µm. (I) Quantification of the mean signal intensity ratio of LRBA or RAB6A signals on LRBA/RAB6A positive tubule vs. nearby LRBA/RAB6A positive Golgi stack. The mean signal intensity of green (endogenous LRBA) or magenta (mScarlet-RAB6A) channels within a 4 × 4 pixels rectangular box over the tubule or Golgi stack was used for calculations. n = 20. (J) HeLa cells stably transfected with AP1M1-mScarlet stained with an antibody against endogenous LRBA. Scale bar 10 µm (upper merged) or 1 µm (lower merged). (K) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and AP4M1-mScarlet or GGA2-mScarlet imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged).

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: NBEA- and AP1M1-positive structures can accumulate endocytosed transferrin. HeLa cells stably transfected with 3xFlag-EGFP-NBEA and AP1M1-mScarlet were imaged live 20 min after addition of 10 µg/ml of AlexaFluor647-Transferrin to the media. Scale bar 10 µm. Playback speed 17x.

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: Characterization of LRBA- and NBEA- positive compartments. (A) HeLa cells stably transfected with 3xFlag-EGFP-NBEA were fixed and stained with an anti-clathrin antibody. Scale bar 10 µm (left merged) or 1 µm (right merged). (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and AP1M1-mScarlet were imaged live with 1 s intervals. NBEA- and AP1M1-positive tubules undergo rapid fission at or close to AP1M1-positive speckles (arrow). Scale bars 1 µm. (C) Live cell imaging of HeLa cells stably transfected with 3xFlag-EGFP-LRBA and AP1M1-mScarlet. LRBA and AP1M1 into distinct subdomains along the length of the growing tubule (arrows). Scale bars 1 µm. (D) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB6A were imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. NBEA-decorated tubules, positive for RAB6A (arrow) do not accumulate transferrin (arrowhead). Scale bars 10 µm (main figures) or 1 µm (zoomed in inserts). (E) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were fixed and stained with anti-Giantin (upper panel) or anti-GF130 (lower panel) antibodies. Endogenous markers of media-Golgi (giantin) and cis-Golgi (GM130) localize in close proximity to perinuclear 3xFlag-EGFP-LRBA positive structures but do not colocalize with LRBA-positive tubules. Scale bars 10 µm (left merged) or 1 µm (right merged).

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Staining, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: LRBA- and NBEA- positive tubules accumulate transferrin starting from 2 min after its addition to the culture media. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and AP1M1-mScarlet were imaged live 10 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Scale bar 10 µm (main figure) or 1 µm (insert magnifications). (B) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were fixed 2 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin and imaged by confocal microscopy. Scale bars 10 µm (left merged) or 1 µm (right merged). (C) Examples of colocalization of stably transfected 3xFlag-EGFP-LRBA and AlexaFluor647-Transferrin, 2 min after addition of transferrin to the culture media. Scale bars 1 µm. (D) HeLa cells stably transfected with 3xFlag-EGFP-NBEA were fixed 3 min after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin and imaged by confocal microscopy. Scale bars 10 µm (left panel) or 1 µm (zoomed-in panels). (E) Quantification of the mean signal intensity ratio of LRBA or transferrin on LRBA/transferrin positive tubule vs. nearby LRBA/transferrin positive vesicle. The mean signal intensity of green (endogenous LRBA) or magenta (endocytosed transferrin, 3 min) channels within a 4 × 4 pixels rectangular box over the tubular or vesicular part of the transferrin-positive compartment was used for calculations. n = 20. (F) Schematic summary of data from . (G) Quantification of the dynamics of colocalization of LRBA or NBEAL2 positive structures and transferrin. HeLa cells stably transfected with 3xFlag-EGFP-LRBA or -NBEAL2 were fixed and imaged at the indicated time points after the addition of 10 µg/ml of AlexaFluor647-Transferrin to the culture media. EGFP or AlexaFluor647 positive structures were segmented and each dot represent the percentage of EGFP-positive objects partially or completely overlapping with AlexaFluor647 positive objects per cell. n ≥ 50 per condition.

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: NBEAL2 shows slower dynamics of colocalization with endocytosed transferrin. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and mScarlet-NBEAL2 were imaged live with 1-min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Tubules positive for LRBA and NBEAL2 undergo fast fusion or fission events (right zoomed-in inserts). LRBA-positive tubules that accumulate transferrin between 2.5 and 6 min after its addition to the media are either negative (arrow) or only weakly positive for NBEAL2 (solid arrowhead). Tubular structures positive for NBEAL2 and transferrin, but negative for LRBA (open arrowheads), could be observed starting from 6.5 min after the addition of transferrin to the culture media (lower zoomed-in inserts) Scale bars 10 µm (main figure) or 1 µm (inset magnifications). (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL2 and mScarlet-LRBA were treated for 30 min with 10 µg/ml of AlexaFluor647-transferrin in complete media, fixed, and imaged by confocal microscopy. LRBA and NBEAL2 localize to opposite ends of the same transferrin-positive tubules. Scale bars 10 µm (main figure) or 1 µm (inset magnifications). (C) Schematic summary of data from .

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: Transferrin does not accumulate in NBEA-positive tubules decorated with TGN markers RAB6A or TGN46. (A) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB4A were imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. Transferrin accumulates in NBEA-positive tubules representing the tubular segment of RAB4A-positive sorting endosomes (arrows). Scale bar 10 µm in the upper row, 1 µm in other panels. (B) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB11A imaged live with 1 min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. NBEA-positive tubules that accumulate transferrin between 2.5 and 6 min after its addition to the media are either negative (open arrowhead) or only weakly positive for RAB11A (arrow). Starting from 9 min 30 s after addition of transferrin, tubules that are positive for RAB11A and transferrin, but negative for NBEA could be detected (solid arrowhead). Scale bar 10 µm in the upper row, 1 µm in other panels. (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and mScarlet-RAB6A were imaged live with 1-min intervals starting 30 s after supplementation of growth media with 10 µg/ml of AlexaFluor647-Transferrin. RAB6A-positive NBEA-decorated tubules (arrow) do not accumulate transferrin (arrowhead). Scale bar: 10 µm in the upper row, 1 µm in other panels. (D) Schematic summary of data from . (E) HeLa cells stably transfected with 3xFlag-EGFP-LRBA were treated for 14 min with 10 µg/ml of AlexaFluor647-transferrin in complete media, fixed, stained with an anti-TGN46 antibody, and imaged by confocal microscopy. LRBA-decorated tubules, positive for the TGN marker TGN46 (arrow) do not accumulate the endocytic recycling cargo transferrin (arrowhead). Scale bars 10 µm (left merged) or 1 µm (right merged).

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Marker

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: The influx of secretory cargo redistributes LRBA and NBEA from recycling to secretory compartments and sequester NBEAL1 and LRBA to different subdomains of secretory tubules. (A) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-mCherry-RUSH were treated for 12 min with 10 µg/ml of AlexaFluor647-Transferrin and 50 µM of biotin to release transport of CTLA4-mCherry-RUSH from the ER, followed by live imaging before and after the treatment. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Golgi-attached LRBA- and CTLA4-RUSH positive secretory tubules lack endocytosed transferrin (arrow) but can acquire transferrin after Golgi detachment (solid arrowhead). LRBA- and transferrin-positive tubules, negative for CTLA4-RUSH are labeled with open arrowheads. Scale bar 10 µm in the upper and middle panel and 1 µm in the lower panel. (B) HeLa cells stably transfected with 3xFlag-EGFP-LRBA and transiently transfected with CTLA4-RUSH-mCherry were imaged before and 12 min after the addition of 50 µM of biotin to the culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (C) HeLa cells stably transfected with 3xFlag-EGFP-NBEA and transiently transfected with CTLA4-RUSH construct imaged before and 12 min after the addition of 50 µM of biotin to culture media. Numbers represent cells not transfected (1) or transfected (2) with CTLA4-RUSH construct before (1 and 2) or 12 min after (1* and 2*) addition of biotin to the media. Scale bars 10 µm (upper two panels) or 1 µm (lower inserts). (D) Schematic summary of data from . (E) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and transiently transfected with CTLA4-RUSH-mCherry were imaged 12 min after the addition of 50 µM of biotin. NBEAL1 decorates the tip of growing Golgi-connected secretory tubules positive for CTLA4-RUSH-mCherry (arrows) that form transport carriers upon tubular fission. Scale bars 1 µm. (F) HeLa cells stably transfected with 3xFlag-EGFP-NBEAL1 and mScarlet-LRBA and transiently transfected with CTLA4-RUSH-mTagBFP2 were imaged live 12 min after the addition of 50 µM of biotin. NBEAL1 and LRBA segregate into different subdomains of Golgi-connected secretory tubules (arrows). mTagBFP2 signal was verified by visual observation, but not imaged, due to the toxicity of the blue-channel imaging setup. Scale bar 10 µm (left merged) or 1 µm (right merged). (G) Schematic summary of data from .

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Stable Transfection, Transfection, Imaging, Labeling, Construct

Journal: The Journal of Cell Biology

Article Title: BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways

doi: 10.1083/jcb.202408173

Figure Lengend Snippet: Chemicals, peptides, and recombinant proteins

Article Snippet: Human Transferrin AlexaFluor647 , Jackson ImmunoResearch , Cat# 009-600-050.

Techniques: Recombinant, Transfection, Isolation, Lysis, Protease Inhibitor

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

doi: 10.3389/fcvm.2022.917238

Figure Lengend Snippet: Effects of mutations in low-density lipoprotein receptor (LDLR) on MT1-MMP-induced LDLR cleavage (A) Diagram of the functional domains of LDLR. LR, ligand binding region. EGF, the epidermal growth factor precursor homology domain. O-linked, the O-linked sugar domain. TM, transmembrane domain. JD, the J.D. mutation (Y807C) identified in a FH patient. C-tail, the C-terminal cytoplasmic tail. (B ) Immunoblotting and (C) quantification. Equal amount of plasmid DNA containing the wild-type (WT) or mutant LDLR deletion cDNA and empty (-) or plasmid containing the wild-type MT1-MMP (MT1) were transfected into HEK293 cells using PEI. 48 h later, whole cell lysate was prepared, and equal amount of total proteins in whole cell lysates was applied to SDS-PAGE and immunoblotting with antibodies as indicated. HA-tagged MT1-MMP and LDLR were detected by a Dylight 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384) and 3143, respectively. The mature and premature forms of the wild type and mutant LDLR were indicated by white arrows and *, respectively. MT1-MMP was indicated by a gray arrow. Actin was detected by a mouse anti-actin monoclonal antibody. (D–F) Biotinylation. HEK293 cells were co-transfected with the WT or mutant LDLR and empty (-) or WT MT1-MMP (MT1) using Lipofectamine 3000. 48 h after, the cells were subjected to biotinylation. Samples were heated at 85 (D,E) or 37°C (F) to elude proteins from Neutravidin beads and then subjected to immunoblotting using a mouse anti-LDLR (HL-1) (D,F) , or a rabbit anti EGFA of LDLR (E) . Actin was detected with a mouse anti-actin antibody. Similar results were observed in three different experiments. Representative images were shown (B,D–F) . Densitometry was determined by a Li-Cor Odyssey Infrared Imaging System. Relative densitometry was defined as the ratio of the densitometry of the mature form of wild-type or mutant LDLR to that of Actin at the same condition (C) . Values were mean ± S.D. of ≥3 experiments. Significance was defined as the mature form of LDLR in the presence of MT1-MMP vs. the mature form of LDLR in the absence of MT1-MMP. *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125).

Techniques: Functional Assay, Ligand Binding Assay, Mutagenesis, Western Blot, Plasmid Preparation, Transfection, SDS Page, Imaging

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

doi: 10.3389/fcvm.2022.917238

Figure Lengend Snippet: Mutations of predicted MT1-MMP cleavage sites on LDLR (A,C) immunoblotting and (B) quantification. HEK293 cells were co-transfected with an equal amount of the wild-type (WT) or mutant LDLR and empty (-) or the wild type MT1-MMP (MT1) plasmid using PEI. 48 h later, whole cell lysate was isolated and applied to immunoblotting. After transfer, the membrane was cut into halves above the 75 kDa protein marker. HA-tagged LDLR on the top membrane was detected with a Dylight™ 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384). m: mature form of LDLR; p: premature form of LDLR. MT1-MMP on the bottom was detected by a Dylight™ 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384). Similar results were observed in three different experiments. Representative images were shown in (A,C) . Densitometry was determined by a Li-Cor Odyssey Infrared Imaging System. Relative densitometry was defined as the ratio of the densitometry of the mature form of wild-type or mutant LDLR to that of Actin at the same condition (B) . Values were mean ± S.D. of ≥3 experiments. Significance was defined as the mature form of LDLR in the presence of MT1-MMP vs. the mature form of LDLR in the absence of MT1-MMP. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125).

Techniques: Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Isolation, Membrane, Marker, Imaging

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

doi: 10.3389/fcvm.2022.917238

Figure Lengend Snippet: Effects of mutations in MT1-MMP on its ability to cleave LDLR (A) Diagram of the functional domains of MT1-MMP. SP, signal peptide. PRO, prodomain. CAT, the catalytic domain. HPX, the hemopexin domain. TM, transmembrane domain. C-Tail, the C-terminal cytoplasmic tail. (B ) Immunoblotting and (C) quantification. Equal amount of plasmid DNA containing the wild-type or mutant MT1-MMP and empty plasmid (-) or the wild-type LDLR-containing plasmid were co-transfected into HEK293 cells using PEI. 48 h later, whole cell lysate was prepared, and equal amount of total proteins in whole cell lysate was applied to immunoblotting. After transfer, the membrane was cut into halves above the 75 kDa protein marker. The top membrane was blotted with a Dylight™ 800-conjugated rabbit anti-HA antibody to recognize HA-LDLR. m: mature form of LDLR; p: premature form of LDLR. The bottom membrane was blotted with a Dylight™ 680-conjugated rabbit anti-HA antibody to detect HA-MT1-MMP (HA-MT1). The bottom part was also blotted with a mouse anti-MT1-MMP antibody to detect MT1-MMP (MT1) (EMD Millipore, MAB3329). TFR, transferrin receptor (TFR). Representative images were shown (B) . Densitometry was determined by a Li-Cor Odyssey Infrared Imaging System. Relative densitometry was defined as the ratio of the densitometry of the mature form (m) of LDLR to that of TFR at the same condition (C) . Values were mean ± S.D. of ≥3 experiments. Significance was defined as the mature form of LDLR in the presence of the wild-type or mutant MT1-MMP vs. the mature form of LDLR in the absence of MT1-MMP (Empty). ns (vs. Empty), no significance, p > 0.05. **** (vs. Empty), p < 0.0001. ### [ΔHPX vs. wild type MT1-MMP (WT)], p < 0.001.

Article Snippet: The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125).

Techniques: Functional Assay, Western Blot, Plasmid Preparation, Mutagenesis, Transfection, Membrane, Marker, Imaging

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

doi: 10.3389/fcvm.2022.917238

Figure Lengend Snippet: Effects of I167A on MT1-MMP trafficking and cell migration (A) Confocal microscopy. HEK293 cells transfected with empty plasmid (Control) or plasmid containing the HA-tagged wild-type or I167A mutant MT1-MMP were subjected to confocal microscopy. MT1-MMP was detected with a rabbit anti-HA polyclonal antibody (Proteintech, 51064-2-AP) and showed in green fluorescence (top and bottom panels). Na + /K + -ATPase was detected by a mouse monoclonal antibody and showed in red fluorescence (middle and bottom panels). Nuclei were visualized with DAPI (blue). An x-y optical section of the cells illustrates the distribution of the wild-type and mutant proteins (magnification: 325X). (B) Transwell assay. HEK293 cells were transfected with empty plasmid (Control) or plasmid containing the HA-tagged wild-type or I167A mutant MT1-MMP using Lipofectamine 3000 and then placed on a collagen type I-coated insert. 48 h after, cells were stained with crystal violet. Cells on the bottom of the insert were imaged, counted, and then divided by the image area. Relative cell numbers were the ratio of the cell numbers of cells transfected with the wild-type or I167A MT1-MMP to that of cells transfected with the empty vector (control), which was defined as 1. Representative images were shown. Values were mean ± S.D. of 3 experiments. The significant difference between two groups (wild type or mutant MT1-MMP vs. the Control) were determined via Student’s t -test. ns (no significance), p > 0.05, ** p < 0.01.

Article Snippet: The following antibodies were used: HL-1, a mouse monoclonal anti-the linker sequence between ligand binding repeat (LR) 4 and LR5 of LDLR antibody ( , ); a rabbit anti-MT1-MMP monoclonal antibody (Abcam, ab51074); a mouse anti-MT1-MMP monoclonal antibody (EMD Millipore, MAB3329); a rabbit anti-HA polyclonal antibody (ProteinTech, 51064-2-AP); a mouse anti-HA monoclonal antibody (ProteinTech, 66006-2-lg); a DylightTM 680-conjugated rabbit anti-HA antibody (Rockland, 600-444-384); a DylightTM 800-conjugated rabbit anti-HA antibody (Rockland, 600-445-384); a mouse anti-actin monoclonal antibody (ProteinTech, 66009-1-Ig); a mouse anti-Na + /K + -ATPase antibody (BD Biosciences, 610993); and a mouse anti-transferrin receptor monoclonal antibody (BD Biosciences, 612125).

Techniques: Migration, Confocal Microscopy, Transfection, Plasmid Preparation, Control, Mutagenesis, Fluorescence, Transwell Assay, Staining