EMBO/soda-nel huggingface dataset:
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 5

Figure 5. Structural basis of Ykt6 double prenylation by GGTase-III.

A Overall view of the central cavity of the β subunit showing insertion of the C-terminal tail of Ykt6. PTAR1 is omitted for clarity. The Cys194-linked geranylgeranyl and Cys195-linked farnesyl moieties are shown in orange and purple, respectively. PPi, inorganic pyrophosphate.

B Electron density of the Cys194-linked geranylgeranyl and Cys195-linked farnesyl moieties (blue mesh; simulated-annealing Fo-Fc omit map contoured at 3σ level).

C, D Detailed views of the geranylgeranyl moiety in the GGTase-III­-product complex (C) and GGPP in the GGTase-III-GGPP complex (D). Residues of the β subunit that form the lipid substrate binding pocket are shown.

E Surface representation of the central cavity of the β subunit showing a tunnel formed near the active site. The Cys195-linked farnesyl moiety is anchored into the hydrophobic tunnel.

F Detailed view of the Cys195-linked farnesyl group bound in the hydrophobic tunnel. Residues of the β subunit that line the hydrophobic tunnel are shown.

G Superposition of the β subunits of FTase (blue), GGTase-I (gray), and GGTase-III (cyan). In FTase and GGTase-I, the hydrophobic tunnel is blocked by the side chain of Leu103 and Ile50, respectively. The corresponding position is replaced by Gly49 in GGTase-III.

H Geranylgeranylation activity of WT GGTase-III and tunnel mutants (100 nM each). Cys195-farnesyl Ykt6ΔAIM (1 μM) and 3H-GGPP (1 μM) were used as substrates (mean ± SEM, n = 3).

I GGPP binding to GGTase-III. WT GGTase-III and tunnel mutants (1 μM each) were incubated with 3H-GGPP (2 μM) at 4ºC for 10 min. After desalting, enzyme-bound 3H-GGPP was quantified by scintillation counting (mean ± SEM, n = 3).

J Alignment of the helix 3 sequences of RabGGTβ orthologues and human FTβ and GGT-Iβ. The position of the conserved glycine residue (Gly49 in human) is highlighted in gray.
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 6

Figure 6. Native Ykt6 is doubly prenylated.

A Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti-Ykt6 antibody. Note the slight difference in the gel mobility of Ykt6 (arrows).

B Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195-farnesyl Ykt6, Cys194/195-diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)-containing buffer and analyzed by immunoblotting with anti-Ykt6 antibody (DOC-PAGE; upper panel). The same samples were analyzed by conventional SDS-PAGE and immunoblotting with anti-Ykt6 antibody (SDS-PAGE; lower panel).

C Prenylation status of Ykt6 in WT and PTAR1 KO cells. Recombinant Ykt6 samples and cell lysates of WT cells, PTAR1 KO cells, and PTAR1 KO cells stably expressing PTAR1 (KO + PTAR1) were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.

D In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37ºC for the indicated times with recombinant GGTase-III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC-PAGE (upper) or SDS-PAGE (lower), and analyzed by immunoblotting with anti-Ykt6 antibody.
A SNARE geranylgeranyltransferase essential for the organization of the Golgi apparatus (2020)
Figure 7

Figure 7. GGTase-III-deficient cells exhibit structural and functional Golgi defects.

A Representative confocal images of the Golgi apparatus in WT or PTAR1 KO HeLa cells. Cells were co-immunostained for GM130 (a cis-Golgi marker) and golgin-97 (a trans-Golgi marker). Images were deconvoluted using Huygens software. The lower panels show magnified images of the boxed region of the upper panels. Scale bars, 5 μm.

B Electron micrographs of the Golgi apparatus in WT or PTAR1 KO HAP1 cells. Arrows indicate Golgi stacks. Arrowheads indicate examples of unfused vesicles accumulated around the swollen Golgi cisternae in PTAR1 KO cells. Scale bars, 500 nm.

C, D Defect in intra-Golgi trafficking in PTAR1 KO cells. (C) VSVG-GFP expressing WT and PTAR1 KO HeLa cells were cultured at 40ºC and then shifted to 32ºC. VSVG-GFP fluorescence images taken at the indicated times after the temperature shift are shown. The right panel shows quantification of VSVG-GFP fluorescence in the Golgi region after the temperature shift (mean ± SEM, n = 6). (D) VSVG-GFP expressing WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were cultured at 40ºC and then shifted to 32ºC. At the indicated time points, cell surface proteins were biotinylated using sulfo-NHS-LC-biotin. Biotinylated VSVG-GFP was purified from cell lysates using avidin agarose and analyzed by immunoblotting with anti-GFP antibody. The right panel shows quantification of the cell surface biotinylated VSVG-GFP (means ± SEM, n = 3). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, ***p < 0.001.

E Analysis of LAMP1 glycosylation. Cell lysates of WT HeLa cells, PTAR1 KO HeLa cells, and PTAR1 KO HeLa cells stably expressing PTAR1 (KO + PTAR1) were analyzed by immunoblotting with anti-LAMP1 antibody (upper). Sialylated LAMP1 was precipitated from the cell lysates using Maackia amurensis leucoagglutinin (MAL) agarose and analyzed by immunoblotting (lower).

F Defect in the Golgi SNARE assembly in PTAR1 KO cells. The Golgi SNARE complex was immunoprecipitated from NEM-treated WT HAP1 cells, PTAR1 KO HAP1 cells, and PTAR1 KO HAP1 cells stably expressing PTAR1 (KO + PTAR1) using control mouse IgG, anti-GS28 IgG, or anti-syntaxin 5 IgG. Immunoprecipitates were analyzed by immunoblotting with antibodies against syntaxin 5, GS28, GS15, and Ykt6. Syntaxin 5 has two isoforms with different translation initiation sites. Inputs were 20% (syntaxin 5, GS28, and GS15) and 2% (Ykt6). The data shown are representative of three independent experiments with similar results.

Expanded View Figure Legends
Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation (2016)
Figure 1

Figure 1. Purification of CBL-OST and CBLB-OST proteins and quantification of their relative abundance.

A CD4+ T cells from wild-type (WT), CBLOST and CBLBOST mice were left unstimulated (-) or stimulated for 2 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin. Eluted proteins were analyzed by immunoblot with antibodies specific for CBLB and CBL. Equal amounts of proteins from whole cell lysates (WCL) were also probed for CBLB and CBL. Note that in the case of CBLB two bands are detected and likely corresponded to the presence of two isoforms differing at their N-terminus (http://www.uniprot.org/uniprot/Q3TTA7). Also shown are loading control corresponding to the same immunoblot probed with anti-CD5, and molecular masses (kDa). Data are representative of at least three experiments.

B CD4+CD8+ thymocytes and mature CD4+ T cells isolated from mice with the specified genotypes were permeabilized and stained with saturating amounts of Strep-Tactin-APC and analyzed by flow cytometry. Background staining was deduced using cells from WT mouse. Data are representative of 2 experiments.
Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation (2016)
Figure 2

Figure 2. Detection of specific interacting partners of CBL and CBLB in peripheral CD4+ T cells prior to and following TCR stimulation.

CD4+ T cells from wild-type (WT), CBLOST and CBLBOST mice were left unstimulated (unstim) or stimulated for 30, 120, 300 or 600 seconds with anti-CD3 and anti-CD4 antibodies. Equal amounts of cell lysates were subjected to affinity purification using Strep-Tactin-Sepharose beads and subjected to MS analysis as described in Material and Methods.

A Histogram comparing the normalized CBL protein intensities resulting from AP-MS analysis of CD4+ T cells from WT and CBLOST mice prior to and after the specified stimulation times. For each time point, the distributions of the normalized intensities of the 9 data points (3 biological replicates x 3 technical replicates) corresponding to CD4+ T from wild-type (WT) and CBLOST mice were compared using the adjusted p-value from a one-way ANOVA test (denoted as p(t)) and the value r(t) corresponding to the enrichment observed in the CBLOST samples as compared to WT samples. Different colors represent distinct biological replicates.

B Proteins are classified as CBL interactors according to their position in a volcano plot in which the mean p-value is plotted against the corresponding mean enrichment for CBLOST versus WT samples. The time point shown corresponds to the unstimulated condition (t=0s). Mean was calculated using bootstrap resampling (see Results and Material and Methods). Proteins that displayed an enrichment r(t) greater than 2-fold and a corresponding p-value p(t) lower than a set threshold (see Material and Methods) in more than 90% of the bootstrap iterations were selected as specific partners (indicated in red). Names of four of the most significantly enriched proteins are indicated. Dashed lines represent thresholds on the p-value p*=10-3 and the enrichment r*=2 used to identify specific interactors of CBL.

C, D List of specific interacting partners of CBLB (C) and CBL (D) ranked according to their maximum mean enrichment across all time points. Heat maps show, for each time point, the fraction of bootstrap iterations for which the corresponding proteins were detected as specific partners (denoted as “P(Detection)”). Red dots indicate the proteins that have been previously reported as CBLB (C) or CBL (D) interactors in the BioGRID database. Ubiquitin (UB) is highlighted with a black arrow.

E Histograms showing the numbers of specific interactors binding to the CBL-OST or CBLB-OST proteins prior to and after TCR stimulation for different times and for which P(Detection) >0.9.

F Pie charts showing the percentages of CBL and CBLB interactors detected in both the present study and the BioGRID public database. The size of the pie charts is proportional to the numbers of interactors per bait.
Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation (2016)
Figure 5

Figure 5. Co-recruitment network predicts the occurrence of physical association between pairs of recruited proteins.

A CBLB first neighbors network within the CBL CN. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. Proteins detected in the CBLB signalosome shown in Fig 3 are colored in red.

B Histograms showing the number and corresponding percentage of CBL interactors also present in the CBLB signalosome according to their distance from CBLB in the CBL CN. The two depicted analyses correspond to the complete and to the high confidence (R>0.8) CNs are depicted.

C Zoom on the high confidence (R>0.8) CRKL and CSK first neighbors subnetwork found within the CBL CN. The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. CSK and CRKL are highlighted in yellow.

D, E, F, G, H Experimental validation of the PPIs predicted on the basis of the high confidence CN shown in C. CD4+ T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in C correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85β and CSK-PI3Kp85β (E) PI3Kp85β-PI3Kp110α, CSK-PI3Kp110α F) CRKL-PI3Kp110α (G). Molecular masses are shown (kDa). Data are representative of at least 2 experiments.

H Summary of the PPIs found in the high confidence CSK and CRKL first neighbors subnetwork (see C) that were validated by co-immunoprecipitation (see D to G). Interactions validated and non validated by co-immunoprecipitation are shown using red and blue edges, respectively. Interactions untractable by co-immunoprecipitation analysis due to the lack of specific antibodies are shown as gray edges.

In C and H, PI3Kp85β and PI3Kp110α are denoted as PIK3R2 and PIK3CA, respectively.
Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation (2016)
Figure 6

Figure 6. Asymmetric functional relationships between CBL and CBLB.

A CD4+ T cells from wild-type (WT), CBLOST and CBLBOST mice were left unstimulated (-) or stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin-Sepharose beads and the resulting protein eluates were analyzed by immunoblotting with anti-ubiquitin (UB), anti-CBLB or anti-CBL antibodies. Also shown are a loading control corresponding to equal amounts of proteins from whole cell lysates (WCL) probed with anti-CD5, and molecular masses (kDa). TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form, likely due to its ubiquitylation (marked with an asterisk). In contrast, no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.

B Representation of the ubiquitin (UB) first neighbors subnetwork within the CBL and CBLB CN. Edge width is coded according to Pearson correlation coefficient (R<0.8 or R>0.8). Red edges depict direct interactions reported in the BioGRID database. Ubiquitin (UB) is highlighted in yellow.

C Immunoprecipitation of CBL from cell lysates of CD4+ T cells isolated from WT or CBLB-deficient mouse (Cblb-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.

D Immunoprecipitation of CBLB from cell lysates of CD4+ T cells isolated from WT or CBL-deficient mouse (Cbl-/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole cell lystates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least 3 experiments.
Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation (2016)
Figure 7

Figure 7. CD5 regulates CBL-mediated ubiquitylation following TCR stimulation in mature CD4+ T cells.

A, B CD4+ T cells from wild-type, Cblb-/- and Cd5-/- mice were stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). CBL (A) and PI3KR1 (PI3Kp85α) (B) were immunoprecipitated from equal amounts of protein lysates. Level of ubiquitylation and amount of CD5 associated proteins were evaluated by immunoblot with antibodies specific for the proteins specified in the left margin. Whole cell lystates (WCLs) were also analyzed with the specified antibodies. Molecular masses are shown (kDa). Data are representative of at least 2 experiments.
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 1

VEGF impairs vascular stabilization despite similar pericyte coverage

A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limb muscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.

B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.

Source data are available online for this figure.
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 2

TGF-β1 and Sema3A are downregulated in vivo by increasing VEGF doses

Muscles were harvested 7 days after implantation of V Low, V Med, and V High clones or control cells (Ctrl).

Gene expression of Pdgfb,Ang1,Ang2,Tgfb1, and Sema3a was quantified by qRT-PCR and expressed as fold-change versus control muscles. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation. TGF-β1: V Low versus V Med P = 0.0047; V Low versus V High P = 0.0379; Sema3A: V Low versus V High P = 0.0042; V Med versus V High P = 0.0179.

Immunohistochemistry for Sema3A on frozen muscle sections confirmed a decreasing protein expression in the areas implanted with myoblasts producing increasing VEGF doses (n = 4). The right panels represent higher-magnification views of the left panels, which encompass the majority of the implantation sites. Scale bar = 50 μm.

In situ hybridization for Sema3A (green) and CD31 (red) mRNA, with nuclei staining by DAPI, on frozen muscle sections. The three lower panels represent a higher-magnification of the area in the white square in the top panel, as a merged image and as individual channels: arrows indicate cells expressing both transcripts (yellow), only Sema3A (green), or only CD31 (red). Scale bar = 30 μm.

Quantification of the percentage of endothelial cells (CD31+) that express Sema3A (left group) and of Sema3A-expressing cells that are endothelial (CD31+, right group). Data represent the mean ± SEM of 12 individual fields of view from three independent muscles (15–94 nuclei/image, 642 total nuclei). No statistics was applied, as data represent complementary sets.

Source data are available online for this figure.
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 3

Increasing VEGF doses inhibit NEM recruitment and endothelial Sema3A expression

Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on cryosections of limb muscles 1 week after injection with V Low, V Med, and V High myoblast clones. Scale bar = 100 μm.

Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis by increasing VEGF doses. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: V Low, n = 48; V Med, n = 39; V High, n = 94; *P < 0.05 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test: V Low versus V Med P = 0.0008; V Low versus V High P < 0.0001; V Med versus V High P = 0.0304.

Endothelial cells (CD31+ quadrant) and NEM (CD11b+ quadrant) were sorted by FACS from muscles 1 week after injection with the same myoblast clones.

Relative gene expression was quantified in the ex vivo FACS-purified endothelial cells (CD31+) and NEM (CD11b+) and expressed as fold-change versus the V High group. Data represent individual values, with mean ± SEM (n = 3–7, as indicated); **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A: V Low versus V High P = 0.0157.

Source data are available online for this figure.
VEGF dose regulates vascular stabilization through Semaphorin3A and the Neuropilin-1+ monocyte/TGF-β1 paracrine axis. (2015)
Figure 4

Sema3A/NRP1 binding is required for NEM recruitment, TGF-β1 upregulation, and Sema3A production

Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated with the anti-NRP1A antibody blocking Sema3A/NRP1 binding (AbαNRP1A) or with control IgG2a. Analyses were performed after 1 week.

A, B Quantification of recruited NEM in sites of new angiogenesis by increasing VEGF doses, expressed as number of CD11b+ cells/mm2 of area (A) or /cm of vessel length (B). Data represent the mean ± SEM of individual images (n) acquired from three muscles/group. (A) Ctrl IgG2a, n = 11; Ctrl AbαNRP1A, n = 17; V Low IgG2a, n = 24; V Low AbαNRP1A, n = 28; V Med IgG2a, n = 25; V Med AbαNRP1A, n = 34; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal–Wallis analysis with Dunn’s multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P = 0.0068; V Low IgG2a versus V Low AbαNRP1A
P = 0.0068; V Med IgG2a versus V Med AbαNRP1A
P = 0.0460. (B) V Low IgG2a versus V Low AbαNRP1A
P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0193; V Med IgG2a versus V Med AbαNRP1A
P = 0.0046.

C Quantification of vessel length density generated by increasing VEGF doses after the different treatments. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG2a, n = 6; Ctrl AbαNRP1A, n = 6; V Low IgG2a, n = 19; V Low AbαNRP1A, n = 20; V Med IgG2a, n = 20; V Med AbαNRP1A, n = 25; *P < 0.05, **P < 0.01 and ***P < 0.001 by Kruskal–Wallis analysis with Dunn’s multiple comparisons test: V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P < 0.0001; V Low IgG2a versus V Med IgG2a P = 0.0014; V Low AbαNRP1A versus V Med AbαNRP1A
P = 0.0179.

D Tgfb1 gene expression in total skeletal muscles after the different treatments, expressed as fold-change versus the Ctrl IgG2a group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); *P < 0.05 and **P < 0.01 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low IgG2a versus Ctrl IgG2a P = 0.0083; V Low IgG2a versus V Low AbαNRP1A
P = 0.0315.

E Immunohistochemistry for Sema3A protein on frozen muscle sections with H&E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 100 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 3; Ctrl IgG2a, n = 23; Ctrl AbαNRP1A, n = 12; V Low IgG2a, n = 53; V Low AbαNRP1A, n = 38; *P < 0.05 and ***P < 0.001 by Kruskal–Wallis analysis with Dunn’s multiple comparisons test: V Low IgG2a versus No stain P = 0.0007; V Low IgG2a versus Ctrl IgG2a P < 0.0001; V Low AbαNRP1A versus Ctrl IgG2a P = 0.0142; V Low IgG2a versus V Low AbαNRP1A
P = 0.0001.

Source data are available online for this figure.