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cd105  (R&D Systems)


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    R&D Systems cd105
    Cd105, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+cd105/pmc13034170-170-28-39?v=R%26D+Systems
    Average 94 stars, based on 30 article reviews
    cd105 - by Bioz Stars, 2026-07
    94/100 stars

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    anti endoglin  (OriGene)
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    (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) <t>using</t> <t>anti-endoglin</t> or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.
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    cd105  (R&D Systems)
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    (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) <t>using</t> <t>anti-endoglin</t> or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.
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    Average 94 stars, based on 1 article reviews
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    cd45 apc  (Elabscience Biotechnology)
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    (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) <t>using</t> <t>anti-endoglin</t> or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.
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    ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for <t>endoglin,</t> and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .
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    cd105  (Cell Signaling Technology Inc)
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    ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for <t>endoglin,</t> and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .
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    555596 pe mouse anti human cd105  (R&D Systems)
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    R&D Systems 555596 pe mouse anti human cd105
    ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for <t>endoglin,</t> and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .
    555596 Pe Mouse Anti Human Cd105, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse cd105  (R&D Systems)
    93
    R&D Systems mouse cd105
    Preparation of mouse BMSCs and their exosomes. (A) Morphological observation of mouse BMSCs at 120× magnification. To ensure objectivity, five random fields were selected for observation in each analysis. (B) Flow cytometry analysis of the expression of surface markers <t>CD105,</t> CD90, CD73 and CD34 in passage 2 BMSCs. (C) SEM characterization of BMSCs‐Exos. (D) Particle size analysis of BMSCs‐Exos using NTA. (E) Western blot analysis of CD9, CD63, and CD81 protein expression in BMSCs‐Exos, with lysis buffer used as a negative control. Three biological replicates were performed for the experiment ( n = 3), and each replicate was tested three times.
    Mouse Cd105, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    antibodies anti mouse cd105  (R&D Systems)
    93
    R&D Systems antibodies anti mouse cd105
    Preparation of mouse BMSCs and their exosomes. (A) Morphological observation of mouse BMSCs at 120× magnification. To ensure objectivity, five random fields were selected for observation in each analysis. (B) Flow cytometry analysis of the expression of surface markers <t>CD105,</t> CD90, CD73 and CD34 in passage 2 BMSCs. (C) SEM characterization of BMSCs‐Exos. (D) Particle size analysis of BMSCs‐Exos using NTA. (E) Western blot analysis of CD9, CD63, and CD81 protein expression in BMSCs‐Exos, with lysis buffer used as a negative control. Three biological replicates were performed for the experiment ( n = 3), and each replicate was tested three times.
    Antibodies Anti Mouse Cd105, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

    Journal: bioRxiv

    Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

    doi: 10.64898/2026.05.12.724648

    Figure Lengend Snippet: (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

    Article Snippet: Mouse monoclonal anti-Flag tag (M2; F1804) was from Millipore Sigma, anti-GAPDH (2118S) from Cell Signaling Technologies (Beverly, MA), anti-VEGFR2 (OTI12C1) from Origene, anti-endoglin (CD-105; NBP2-22122) and anti-LAMP2 (NBP2-22217) from Novus Biologicals, Inc, anti-Rab5 (D-11) and anti-Rab7 (B-3) from Santa Cruz Biotechnology, Inc. Chicken polyclonal anti-GFAP (ab4674) was from Abcam.

    Techniques: Mutagenesis, Generated, Software, Injection, Isolation, Immunoprecipitation, Control, Western Blot, Marker, Staining, Fluorescence

    (A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

    Journal: bioRxiv

    Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

    doi: 10.64898/2026.05.12.724648

    Figure Lengend Snippet: (A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

    Article Snippet: Mouse monoclonal anti-Flag tag (M2; F1804) was from Millipore Sigma, anti-GAPDH (2118S) from Cell Signaling Technologies (Beverly, MA), anti-VEGFR2 (OTI12C1) from Origene, anti-endoglin (CD-105; NBP2-22122) and anti-LAMP2 (NBP2-22217) from Novus Biologicals, Inc, anti-Rab5 (D-11) and anti-Rab7 (B-3) from Santa Cruz Biotechnology, Inc. Chicken polyclonal anti-GFAP (ab4674) was from Abcam.

    Techniques: Mutagenesis, Control, Expressing, Staining, Software

    ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for endoglin, and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for endoglin, and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Purification, Staining, SDS Page, Western Blot, Control, Marker, Incubation, Knockdown, Transfection

    ( A ) Nanoparticle tracking analysis traces for B16F1 control (shScr) and endoglin-KD (shEng) SEVs. ( B ) SEV secretion rates from B16F1 shEng stable lines. N=5 biological replicates. ( C ) Representative western blot of Endoglin-KD in transient siRNA-transfected B16F1 cells. ( D ) Filopodia numbers in siRNA-transfected B16F1 cells (≥23 cells per condition per biological replicate, from three biological replicates). ( E ) Images from control and shHrs cells incubated with purified EV, corresponding to graph in . Scale bar in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) Nanoparticle tracking analysis traces for B16F1 control (shScr) and endoglin-KD (shEng) SEVs. ( B ) SEV secretion rates from B16F1 shEng stable lines. N=5 biological replicates. ( C ) Representative western blot of Endoglin-KD in transient siRNA-transfected B16F1 cells. ( D ) Filopodia numbers in siRNA-transfected B16F1 cells (≥23 cells per condition per biological replicate, from three biological replicates). ( E ) Images from control and shHrs cells incubated with purified EV, corresponding to graph in . Scale bar in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Control, Western Blot, Transfection, Incubation, Purification

    ( A ) Western blot of Endoglin KD in HT1080 cells. ( B ) Nanoparticle tracking analysis traces of SEVs purified from shScr and shEng HT1080 cells showing size distribution (diameter) of SEVs and particles/mL/cell (N=3 biological replicates). ( C ) SEV secretion rates of HT1080 shScr and shEng HT1080 cells. ( D ) Representative images of HT1080 shScr and shEng cells. Images have been edited with brightness and contrast for ease of visibility. Scale bar in wide field and zoom insets = 10 mm. ( E ) Quantitation of filopodia density for control and shEng HT1080 cells.≥20 cells per condition per biological replicate, from four biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) Western blot of Endoglin KD in HT1080 cells. ( B ) Nanoparticle tracking analysis traces of SEVs purified from shScr and shEng HT1080 cells showing size distribution (diameter) of SEVs and particles/mL/cell (N=3 biological replicates). ( C ) SEV secretion rates of HT1080 shScr and shEng HT1080 cells. ( D ) Representative images of HT1080 shScr and shEng cells. Images have been edited with brightness and contrast for ease of visibility. Scale bar in wide field and zoom insets = 10 mm. ( E ) Quantitation of filopodia density for control and shEng HT1080 cells.≥20 cells per condition per biological replicate, from four biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Western Blot, Purification, Quantitation Assay, Control

    ( A ) Western blot showing b1-integrin, TGFb1, ALK1 levels in control (shScr) and endoglin-KD (shEng) B16F1 SEVs. ( B ) Filopodia density analysis of B16F1 shScr and shEng cells treated with BMP-9.≥20 cells per condition per biological replicate, from three biological replicates. ( C ) Filopodia density analysis of B16F1 shScr and shEng cells treated with TGFb1.≥20 cells per condition per biological replicate, from three biological replicates. ( D ) Filopodia density analysis of B16F1 shScr and shEng cells plated on 20 µg/ml fibronectin (FN) or 100 µg/mL poly-D-lysine (PDL). ≥20 cells per condition per biological replicate, from three biological replicates. ( E ) Filopodia density analysis of B16F1 shScr and shEng cells plated on PDL or 2 µg/mL rhTHSD7A for the indicated time points, then fixed and stained for filopodia. ≥20 cells per condition per biological replicate, from biological replicates. Figure 6—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) Western blot showing b1-integrin, TGFb1, ALK1 levels in control (shScr) and endoglin-KD (shEng) B16F1 SEVs. ( B ) Filopodia density analysis of B16F1 shScr and shEng cells treated with BMP-9.≥20 cells per condition per biological replicate, from three biological replicates. ( C ) Filopodia density analysis of B16F1 shScr and shEng cells treated with TGFb1.≥20 cells per condition per biological replicate, from three biological replicates. ( D ) Filopodia density analysis of B16F1 shScr and shEng cells plated on 20 µg/ml fibronectin (FN) or 100 µg/mL poly-D-lysine (PDL). ≥20 cells per condition per biological replicate, from three biological replicates. ( E ) Filopodia density analysis of B16F1 shScr and shEng cells plated on PDL or 2 µg/mL rhTHSD7A for the indicated time points, then fixed and stained for filopodia. ≥20 cells per condition per biological replicate, from biological replicates. Figure 6—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Western Blot, Control, Staining

    ( A ) Western blot analysis of total cell lysates (TCL) and SEVs from HT1080 control and shEng cells +/-rescue with WT endoglin or control expression vectors. The figure was made from cropped images of membranes to remove irrelevant lanes. ( B ) Quantification of endoglin expression (normalized to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( C ) Quantification of THSD7A expression (relative to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( D ) Quantification of filopodia in HT1080 control cells and shEng cells rescued with WT endoglin expression. N=3, at least 30 total cells per condition. ( E ) Representative confocal images of THSD7A-mScarlet-expressing control and shEng HT1080 cells immunostained for CD63. Box 1 shows extracellular THSD7A and CD63 deposits. Box 2 shows intracellular CD63-positive MVEs. For both boxes, the zoomed images have been adjusted for brightness and contrast (to equivalent levels for control and shEng cells) for easy visualization. Note that the overlap of THSD7A (magenta) and CD63 (green) gives a white signal, pointed out with white arrowheads in the shEng merged image in Zoom 2. Scale bar is 10 mm in wider field view and 5 mm in zoom insets. ( F ) Quantification of colocalization of internal CD63 and mScarlet signals in HT1080 cells from nonadjusted images.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 7—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 7—source data 2. Original files for western blot analysis displayed in .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) Western blot analysis of total cell lysates (TCL) and SEVs from HT1080 control and shEng cells +/-rescue with WT endoglin or control expression vectors. The figure was made from cropped images of membranes to remove irrelevant lanes. ( B ) Quantification of endoglin expression (normalized to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( C ) Quantification of THSD7A expression (relative to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( D ) Quantification of filopodia in HT1080 control cells and shEng cells rescued with WT endoglin expression. N=3, at least 30 total cells per condition. ( E ) Representative confocal images of THSD7A-mScarlet-expressing control and shEng HT1080 cells immunostained for CD63. Box 1 shows extracellular THSD7A and CD63 deposits. Box 2 shows intracellular CD63-positive MVEs. For both boxes, the zoomed images have been adjusted for brightness and contrast (to equivalent levels for control and shEng cells) for easy visualization. Note that the overlap of THSD7A (magenta) and CD63 (green) gives a white signal, pointed out with white arrowheads in the shEng merged image in Zoom 2. Scale bar is 10 mm in wider field view and 5 mm in zoom insets. ( F ) Quantification of colocalization of internal CD63 and mScarlet signals in HT1080 cells from nonadjusted images.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 7—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 7—source data 2. Original files for western blot analysis displayed in .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Western Blot, Control, Expressing

    Control and endoglin-KD HT1080 cells were plated on coverslips coated with poly-D-lysine (PDL) or THSD7A. In some cases, cells were treated with the Cdc42 inhibitor ML141 (10 µM) or transfected with the dominant active Cdc42 mutant Q61L, as indicated.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: Control and endoglin-KD HT1080 cells were plated on coverslips coated with poly-D-lysine (PDL) or THSD7A. In some cases, cells were treated with the Cdc42 inhibitor ML141 (10 µM) or transfected with the dominant active Cdc42 mutant Q61L, as indicated.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Control, Transfection, Mutagenesis

    ( A ) In tumor cells, endoglin and THSD7A are trafficked into intralumenal vesicles (ILV) in multivesicular endosomes (MVEs) for secretion. Inhibiting the exosome biogenesis pathway by blocking Hrs or inhibiting MVE docking by blocking Rab27a subsequently reduces exosome secretion and filopodia formation. SEVs carrying THSD7A can induce filopodia on target cells via Cdc42, leading to increased cell motility and metastasis. When endoglin levels are lowered (such as by KD), THSD7A is retained inside cells in CD63-positive endosomes, and its levels in SEVs are greatly decreased. The drop in THSD7A levels in endoglin-KD EVs could be due either to a lack of trafficking into ILVs or, alternatively, enhanced lysosomal degradation of THSD7A-containing MVEs. Given that THSD7A accumulates in CD63-positive endolysosomal compartments in endoglin-KD cells , the latter possibility seems more likely. The cartoon was created using BioRender.com . ( B ) In primary neurons, exosome biogenesis is controlled by the formation of ILVs by Hrs and MVE docking is controlled by Rab27b. Knockdown of either of these proteins results in reduced formation of filopodia, dendritic spines, and synapses in both cortical and hippocampal neurons. Similar to cancer cells, THSD7A is carried in neuronal SEVs and induces filopodia. The cartoon was created using BioRender.com .

    Journal: eLife

    Article Title: Secreted exosomes induce filopodia formation

    doi: 10.7554/eLife.101673

    Figure Lengend Snippet: ( A ) In tumor cells, endoglin and THSD7A are trafficked into intralumenal vesicles (ILV) in multivesicular endosomes (MVEs) for secretion. Inhibiting the exosome biogenesis pathway by blocking Hrs or inhibiting MVE docking by blocking Rab27a subsequently reduces exosome secretion and filopodia formation. SEVs carrying THSD7A can induce filopodia on target cells via Cdc42, leading to increased cell motility and metastasis. When endoglin levels are lowered (such as by KD), THSD7A is retained inside cells in CD63-positive endosomes, and its levels in SEVs are greatly decreased. The drop in THSD7A levels in endoglin-KD EVs could be due either to a lack of trafficking into ILVs or, alternatively, enhanced lysosomal degradation of THSD7A-containing MVEs. Given that THSD7A accumulates in CD63-positive endolysosomal compartments in endoglin-KD cells , the latter possibility seems more likely. The cartoon was created using BioRender.com . ( B ) In primary neurons, exosome biogenesis is controlled by the formation of ILVs by Hrs and MVE docking is controlled by Rab27b. Knockdown of either of these proteins results in reduced formation of filopodia, dendritic spines, and synapses in both cortical and hippocampal neurons. Similar to cancer cells, THSD7A is carried in neuronal SEVs and induces filopodia. The cartoon was created using BioRender.com .

    Article Snippet: Antibody , anti-mouse Endoglin (Rabbit polyclonal) , Cell Signaling , 3290 , WB 1:1000.

    Techniques: Blocking Assay, Knockdown

    Preparation of mouse BMSCs and their exosomes. (A) Morphological observation of mouse BMSCs at 120× magnification. To ensure objectivity, five random fields were selected for observation in each analysis. (B) Flow cytometry analysis of the expression of surface markers CD105, CD90, CD73 and CD34 in passage 2 BMSCs. (C) SEM characterization of BMSCs‐Exos. (D) Particle size analysis of BMSCs‐Exos using NTA. (E) Western blot analysis of CD9, CD63, and CD81 protein expression in BMSCs‐Exos, with lysis buffer used as a negative control. Three biological replicates were performed for the experiment ( n = 3), and each replicate was tested three times.

    Journal: The FASEB Journal

    Article Title: Bone Marrow Mesenchymal Stem Cell‐Derived Exosomes Promote M2 Polarization and Protect Against Acute Lung Injury

    doi: 10.1096/fj.202502772R

    Figure Lengend Snippet: Preparation of mouse BMSCs and their exosomes. (A) Morphological observation of mouse BMSCs at 120× magnification. To ensure objectivity, five random fields were selected for observation in each analysis. (B) Flow cytometry analysis of the expression of surface markers CD105, CD90, CD73 and CD34 in passage 2 BMSCs. (C) SEM characterization of BMSCs‐Exos. (D) Particle size analysis of BMSCs‐Exos using NTA. (E) Western blot analysis of CD9, CD63, and CD81 protein expression in BMSCs‐Exos, with lysis buffer used as a negative control. Three biological replicates were performed for the experiment ( n = 3), and each replicate was tested three times.

    Article Snippet: For flow cytometric analysis, cells were fixed with 1% methanol, blocked with 1% BSA and 0.1% goat serum, incubated with antibodies anti‐mouse CD105, CD90, CD73 and CD34 (ab314950/ab25322/FAB4488V/ab23830; Abcam, Cambridge, UK or R&D Systems, MN, USA) each diluted 1:250, then washed and resuspended in Dulbecco's Phosphate‐Buffered Saline (dPBS).

    Techniques: Flow Cytometry, Expressing, Particle Size Analysis, Western Blot, Lysis, Negative Control