Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were either treated with 0.1 μg/mL PMA or 5 μM ionomycin (Iono) for 30 min, or infected with CLJ1 (90 min), or left untreated/uninfected (NI). Cellular extracts were analysed by Western blot using E-cadherin and β-actin antibodies. FL, full-length; CTF, C-terminal fragment. The experiment was performed twice. B . A549 cells (left) or HUVECs (right) were pre-treated with DMSO, the general metalloprotease inhibitor GM6001 (10 μg/mL) or the specific ADAM10 inhibitor GI254023X (5 μM) and then incubated with CLJ1 or IHMA87 (90 min), or uninfected (NI). Cellular extracts were analysed as above. The experiment was performed twice for A549 and 3 times for HUVECs. C . A549 or ADAM10-deficient A549 (A549 ADAM10 -/- ) cells were incubated with either CLJ1 or IHMA87. Cellular extracts were prepared at different time points post-infection as indicated and analysed by Western blot (left). The right panel shows the FACS analysis of ADAM10 surface expression of both cell lines, as well as the negative control. The experiment was performed 3 times. D . Similar experiment with HUVECs, either transfected with ADAM10 siRNA or untreated. The experiment was performed twice.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Infection, Western Blot, Incubation, Expressing, Negative Control, Transfection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: Plasma membrane rupture was monitored by LDH release in the supernatant. A549 or A549 ADAM10 -/- cells were incubated for 5 hours with IHMA87, IHMA87Δ exlA or IHMA87Δ exlA/exlA strains. The supernatants were the tested for LDH activity. The histograms show the mean ± s.d. of triplicates. The data are representative of 3 experiments.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Incubation, Activity Assay

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were incubated with various concentrations of TFP, as indicated, to impede calmodulin interaction with ADAM10. Ionomycin was used as positive controls. E-cadherin cleavage was assessed by Western blot. The experiment was performed twice. B . Western blot analysis of A549 E-cadherin contents after infection with CLJ1 or IHMA87, in presence or absence of BAPTA-AM. Both experiments were performed 3 times. C . LDH release of A549 cells infected with either CLJ1 or IHMA87, in presence/ absence of BAPTA-AM. Student’s t-test showed significance between the two treatments for both CLJ1 and IHMA87 data (p-values indicated above the bars). The experiment was performed 3 times.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Western Blot, Infection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells (left) or HUVECs (right) were incubated with the S . marcescens ShlA-secreting strain Db11, or with the non-ShlA-secreting mutant 21C4. Cellular extracts were analysed for their E- or VE-cadherin contents. The experiment was performed twice for the left panel and once for the right panel. B . Similar analysis using A549 ADAM10 -/- . The experiment was performed once. C . Similar analysis using A549 cells, in presence/ absence of BAPTA-AM. D-G . Intracellular Ca 2+ contents and plasma membrane permeability were measured using Fluo3-AM and Draq7 fluorescent probes, respectively. A549 cells ( D,F ) and HUVECs ( E,G ) were infected with Db11 ( D,E ) or 21C4 ( F,G ) and fluorescence was recorded on both channels by videomicroscopy. Five cells were analysed in each case; the Fluo3 intensities are represented by straight lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. Data are representative of 8 and 5 independent experiments for A549 and HUVECs, respectively.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Mutagenesis, Clinical Proteomics, Membrane, Permeability, Infection, Fluorescence

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: In uninfected cells, pro-ADAM10 is associated with calmodulin, preventing its maturation and export to the plasma membrane. Pore formation by ExlA or ShlA induces a massive Ca 2+ influx in host cells. Intracellular Ca 2+ interacts with the Ca 2+ -binding protein calmodulin, which detaches from pro-ADAM10, allowing its maturation to m-ADAM10. m-ADAM10 cleaves E- and VE-cadherin in epithelial and endothelial cells, respectively, provoking intercellular junction rupture.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Binding Assay

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 1. Specificity of a-ADAM10 monoclonal antibodies. (A) Alignment of mouse, human and bovine ADAM10 cysteine-rich domain sequences (AA 551– 646). In the human and bovine sequences, only residues not homologous to mouse are shown. (B) Comparison of binding of mouse hybridoma (fusion) and isolated cell clone supernatants to serially diluted, immobilised bovADAM10 ECD by ELISA. Binding of non-immunised mouse serum (control) is shown for comparison. (C) Binding of endogenous huADAM10 by a-ADAM10 hybridoma clones, or the R&D ADAM10 mAb 1427, was compared by immunoprecipitation from equivalent HEK293 cell lysates and western blotting with an a-ADAM10 pAb; u, unprocessed; p, processed ADAM10. (D) The specificity of 8C7 for ADAM10 was tested by immunoprecipitation from lysates of ADAM10 knockout (2/2) and Wt (+/+) mouse embryonic fibroblasts (MEFs), and a-ADAM10 pAb western blot.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Bioprocessing, Comparison, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Control, Clone Assay, Immunoprecipitation, Western Blot, Knock-Out

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 2. Co-staining of cells with ADAM10 mAb 8C7 and ephrin-A5-Fc reveals colocalisation and co-internalisation with EphA3. (A) EphA3/ HEK293 cells were incubated on ice with Alexa647–8C7 mAb and fixed for imaging (0 min) or first allowed to warm to 37˚C for 60 min. (B) Cells were labelled with Alexa647–8C7 and with Alexa488–ephrin-A5-Fc and fixed immediately (0 min) or incubated at 37˚C with a-humanFc to cluster ephrin- A5-Fc for the indicated time periods before fixation. The insets are enlarged images of the regions within the dotted lines. Cells incubated for 60 min with Alexa488–ephrin-A5-Fc alone are shown as a control in the bottom panels. Scale bars: 25 mm.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Staining, Incubation, Imaging, Control

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 3. Site-directed mutagenesis of the ADAM10 substrate-binding pocket disrupts mAb binding. (A) Structure of the bovine ADAM10 D and C domains showing the location of key residues targeted by site-directed mutagenesis. (B) Comparison of aADAM10 mAb binding to Wt and substrate-binding pocket mutant huADAM10. Alanine substitutions at Glu 573, 578 and 579 (3EA) or at residues 617 and 618 (617AA) were made in huADAM10-GFP, and Wt and mutant constructs were transfected into ADAM102/2 MEFs (control: untransfected). Binding of a-ADAM10 mAbs was assessed by immunoprecipitation from equivalent cell lysates, and western blotting with a-ADAM10 pAb (non-relevant lanes removed; the altered molecular mass pattern reflects the GFP-tagged huADAM10). The graph shows binding of 8C7 and 3A8 relative to the R&D mAb, determined by densitometry (one-way ANOVA; **P,0.01 compared to R&D sample; n.s., not significant; n53).

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Mutagenesis, Binding Assay, Comparison, Construct, Transfection, Control, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 5. ADAM10 mAb 8C7 inhibits EphA3 phosphorylation in response to stimulation by cell-bound ephrin. (A) 293/EphA3 cells were pretreated with 0, 10 and 100 mg/ml of 8C7 mAb for 2 h and stimulated for the indicated times. a-EphA3 immunoprecipitates from the cell lysates were analysed by western blot with a-phosphotyrosine (pY) and a-EphA3 antibodies as indicated. A representative image from four experiments is shown. (B) EphA3 phosphorylation relative to EphA3 protein levels was calculated from replicate experiments as described in A, using densitometry analysis. Graph shows means 6 s.e.m., n54. (C) 8C7 does not inhibit EphA3 phosphorylation induced by soluble clustered ephrin-A5. EphA3/293 cells, pre-incubated with or without 8C7 (100 mg/ml) for 2 hours, were stimulated for 20 min with pre-clustered ephrin-A5-Fc, or left unstimulated, as indicated. EphA3 immunoprecipitates from cell lysates were analysed by western blotting as in A.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 6. ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell repulsion. (A) EphB2/HEK293 cells labelled with Cell Tracker Green were pre-treated with vehicle (Cont), 8C7 (50, 200 or 400 mg/ml), or with GM6001 (GM, 50 mM), and plated onto coverslips pre-coated with fibronectin and stripes of alexa594-labelled ephrin-A5-Fc. As a comparison, cells expressing a signalling-deficient EphB2 mutant (DICD) were also used. After 18 hours the cells were imaged by fluorescence microscopy, from which examples are shown (8C7, 400 mg/ml). Scale bar: 250 mm. (B) The percentage of cells adhering to ephrin stripes was calculated from ,20 images for each treatment; the graph shows the averages 6 s.e.m. from three experiments. (C) 8C7 inhibits ephrin-A5-induced EphB2 phosphorylation. Effects of 8C7 treatment on activation of EphB2/HEK293 cells by ephrin-A5/HEK293 cells was assessed as in Fig. 5A, following stimulating for 40 minutes.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Comparison, Expressing, Mutagenesis, Fluorescence, Microscopy, Phospho-proteomics, Activation Assay

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: The mature membrane form of ADAM10 is expressed on HL cells. Panels (A), (B), (D) Lysates obtained from HL LN cell suspensions (A) or HL cell lines (B) or LN MSCs obtained by culturing LN cell suspensions from HL patients (D) were subjected to Western blot as described in Materials and Methods; membranes were probed with the anti-ADAM10 or anti-β actin mAb followed by the relevant HRP-conjugated secondary antibodies and developed with the HRP substrate. In each blot, the precursor form (p) and the mature form (m) of ADAM10 molecule is indicated. Panels (C) and (E) Surface expression of ADAM10 on KMH2, L540, L428 (Ca, Cb, Cc, dark gray histograms) or MSC773 or RS773 (Ea, Eb) was evaluated with the specific mAb directed against the mature form of ADAM10 followed by APC-conjugated GAM and FACS analysis; results are expressed as Log far red fluorescence intensity, a.u., vs. number of cells.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Membrane, Western Blot, Expressing, Fluorescence

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: ADAM10 silencing leads to decreased shedding of NKG2D-L. KMH2 and L428 cells were transfected with ADAM10 (siADAM10) or ADAM17 (siADAM17) siRNA or non-targeting siRNA (siNT) pool as negative control (KMH2: panels A, B; L428: panels C, D). Protein expression was analyzed by Western blot (panels Aa and Ca), and FACS analysis (Ab and Cb; in each histogram the percentage and MFI of positive cells is shown) with the specific anti-ADAM10 or anti-ADAM17 antibodies, 72 h after transfection. Soluble MIC-B (Ba, Da) or ULBP3 (Bb, Db) or sALCAM (Bc, Dc) were evaluated by ELISA in SN (collected upon further 24 h of culture 72 h after transfection). Results in (B) and (D) are expressed as pg/mL/10 5 cells and are the mean ± SD from three independent experiments. * p <0.001 vs siNT.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Negative Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: In vitro enzymatic activity (IC 50 nM values) a of new compounds LT4 and MN8 and the reference compounds JG26 and GI254023X.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: In Vitro, Activity Assay

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: ADAM10 inhibitors reduce the shedding of NKG2D-L by HL cell lines and maintain the binding of NKG2D receptor. L428 cells were exposed to culture medium alone, DMSO or GI254023X (GIX), JG26, MN8 or LT4 (at 10 to 2.5 μM concentration) for 24 h (panel A), followed by 100 μM Na 3 VO 4 as pervanadate for 40 min at 37°C (panel B and C). Then, SN were harvested and sMIC-A (Aa, Ba), sMIC-B (Ab, Bb), sULBP2 (Ac, Bc), sULBP3 (Ad, Bd) or sALCAM (C) measured by specific ELISA. Results are expressed as ng/mL/10 6 cells and are representative of four independent experiments. * p <0.001 vs. DMSO. Panels (D) and (E) L428 cells exposed for 24 h to DMSO or 10 μM LT4 or 100 μM Na 3 VO 4 as pervanadate, in the absence or presence of 10 μM LT4 as indicated, were harvested and evaluated for the expression of ULBP2 (D) with the specific mAb followed by APC-conjugated GAM or for the binding of the chimeric receptor (FcNKG2D, panel E) followed by APC-conjugated anti-human Fc antiserum, by FACS analysis; results are expressed as Log far red fluorescence intensity (arbitrary units, a.u.) vs. number of cells. In each subpanel: percentage and mean fluorescence intensity (MFI, a.u.) of positive cells. One representative experiment out of four.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: Exposure to ADAM10 inhibitors increases the sensitivity of HL cell lines to NKG2D-dependent cell killing. Cytolytic activity of NK cells (n = 6, panel A and B) or γδ T cells (n = 6, panel C) was analyzed against L428 (Aa, Ab, Ca) or L540 (Ba, Bb, Cb) cell lines at E:T ratio of 10:1 in a 4-h 51 Cr-release assay. Some samples were set up after exposure of the target cell lines to either medium, or DMSO or LT4 or MN8 (Aa, Ba, Ca, Cb), GIX or JG26 (Ab, Bb) at 10 μM concentration for 24 h. In some samples, effector cells were exposed to saturating amounts (5 μg/mL) of the anti-NKG2D mAb at the onset of the cytotoxicity assay; an unrelated mAb, matched for the isotype, used as control, did not exert any effect (not shown). Results are expressed as % specific lysis calculated as described in Materials and Methods.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Release Assay, Concentration Assay, Cytotoxicity Assay, Control, Lysis

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: Improvement of HL cell lysis by exposure to ADAM10 inhibitor LT4 and anti-TGFβ. Panel (A) NKG2D expression before (upper histograms) or after treatment with TGFβ (10 ng/mL), (middle histograms) or with TGFβ and anti-TGFβ mAb (1 µg/mL), on NK cells (Aa) or γδ T cells (Ab). In each subpanel: percentage of positive cells and MFI (a.u.). Panel (B) Cytolytic activity of NK cells (Ba) orγδ T cells (Bb) was analyzed against L428 cell line at E:T ratio of 5:1 in a 4-h 51 Cr-release assay. Some samples were set up after exposure of the target cell lines to LT4 or MN8 at 10 μM concentration for 24 h. To some samples, we added effector cells exposed to TGFβ (10 ng/mL), with or without saturating amounts (1 μg/mL) of the anti-TGFβ mAb, as indicated. Results are expressed as % inhibition or stimulation of specific lysis calculated as described in Materials and Methods. * p <0.001 vs. TGFβ. ** p <0.001 vs. TGFβ + anti-TGFβ. # p <0.001 vs. TGFβ + anti-TGFβ on untreated L428 cells.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Lysis, Expressing, Activity Assay, Release Assay, Concentration Assay, Inhibition

Journal: Journal of Clinical Investigation

Article Title: Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis

doi: 10.1172/jci168450

Figure Lengend Snippet: Figure 1. Staphylococcus aureus targets endothelial ADAM10 to cause lethal sepsis. (A) ADAM10 (red) staining in the descending aorta and mesenteric artery from control or VE-Cad ADAM10–/– mice. DAPI (blue) denotes cell nuclei; collagen autofluorescence (green). Scale bars: 30 μm. (B) Survival following lethal S. aureus infection in VE-Cad ADAM10–/– (n = 37) or control (n = 18) female mice. Independent experiments were repeated 4 times, and data were pooled. (C) Survival following lethal purified Hla sepsis in VE-Cad ADAM10–/– (n = 9) or control (n = 10) female mice. Indepen- dent experiments were repeated twice, and data were pooled. (D) Serum IL-10 analysis 8 and 24 hours after infection in female VE-Cad ADAM10–/– mice or controls. Data represent 3 independent pooled experiments. Data are presented as the mean ± SEM. (E) Mouse platelet count enumerated 4 hours after lethal infection with S. aureus in male and female mice. Data are from 3 independent pooled experiments and are presented as the mean ± SD. *P ≤ 0.05 and **P ≤ 0.01, by unpaired, 2-tailed t test.

Article Snippet: Vessels were then incubated in blocking buffer (PBS with 3% BSA, 1% fish gelatin, 0.5% Triton X-100) for 1 hour followed by a primary anti–mouse Adam10 ectodomain antibody (5 μg/mL, R&D Systems, AB946) at 4°C overnight and then a secondary antibody, Alexa Fluor 594 donkey anti-goat (1:500, Life Technologies, Thermo Fisher Scientific, A11058) for 1 hour at room temperature.

Techniques: Staining, Control, Infection, Purification

Journal: Journal of Clinical Investigation

Article Title: Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis

doi: 10.1172/jci168450

Figure Lengend Snippet: Figure 2. ADAM10 alters endothelial cell–platelet interactions in response to S. aureus and contributes to sepsis-associated injury. (A) Representative 2-photon image of control or VE-Cad ADAM10–/– mouse livers 6 to 8 hours after nonlethal S. aureus sepsis. Vasculature (red, Qdots655), platelets (green, GPIbβ). Scale bars: 50 μm. Lower panels display images outlined by a dashed line box. Scale bars: 20 μm. White arrows denote thrombi. (B) Quantification of the total area of platelet accumulation within the vasculature in mouse liver as treated in A. Data represent 5–7 FOV per mouse in control (n = 7F) and VE-Cad ADAM10–/– (n = 6F) mice. Data are presented as the mean ± SEM. Data for individual FOV within each mouse are shown in Supplemental Figure 2A. (C) Representative images of the liver 24 hours after S. aureus infection in control or VE-Cad ADAM10–/– mice. (D) H&E-stained liver sections from control or VE-Cad ADAM10–/– mice 24 hours after infection, with the area of necrosis outlined. Scale bars: 100 μm. Images in C and D are representative of 5 mice per condition from 2 independent experiments. (E) Serum ALT in infected control (n = 11 males, 7 females) or VE-Cad ADAM10–/– (n = 7 males, 6 females) mice 24 hours after nonlethal S. aureus infection. Data represent 3 independent pooled experiments and are presented as the mean ± SEM. (F) Representative 2-photon images of control or VE-Cad ADAM10–/– mouse livers 2–4 hours after lethal S. aureus sepsis. Vasculature (red, Qdots655); vWF (green). Scale bars: 10 μm. White arrows denote vWF deposition. (G) Quantification of the total area of vWF accumulation within the vasculature in mouse liver as treated in F. Data represent 5–6 FOV per mouse in control (n = 6F) and VE-Cad ADAM10–/– (n = 4 females, 2 males) mice per group and represent the mean ± SEM. Data for individual FOV within each mouse are shown in Supplemental Figure 3F. *P ≤ 0.05 and **P ≤ 0.01, by nested t test for in vivo imaging (B and G) or unpaired, 2-tailed t test (E).

Article Snippet: Vessels were then incubated in blocking buffer (PBS with 3% BSA, 1% fish gelatin, 0.5% Triton X-100) for 1 hour followed by a primary anti–mouse Adam10 ectodomain antibody (5 μg/mL, R&D Systems, AB946) at 4°C overnight and then a secondary antibody, Alexa Fluor 594 donkey anti-goat (1:500, Life Technologies, Thermo Fisher Scientific, A11058) for 1 hour at room temperature.

Techniques: Control, Infection, Staining, In Vivo Imaging

Journal: Journal of Clinical Investigation

Article Title: Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis

doi: 10.1172/jci168450

Figure Lengend Snippet: Figure 3. Endothelial ADAM10 mediates lethal sepsis in a pathogen-specific manner. Survival curves for control and VE-Cad ADAM10–/– mice infected with lethal P. aeruginosa (A, n = 21 [13 males, 8 females], n = 20 [11 males, 8 females]); S. pneumoniae (B, n = 24 [8 males, 16 females], n = 23 [11 males, 12 females]; GBS (C, n = 23 [9 males, 14 females], n = 14 [7 males, 7 females]; or C. albicans (D, n = 12 males, 13 males ). **P ≤ 0.01, by log-rank (Mantel-Cox) test for survival curves.

Article Snippet: Vessels were then incubated in blocking buffer (PBS with 3% BSA, 1% fish gelatin, 0.5% Triton X-100) for 1 hour followed by a primary anti–mouse Adam10 ectodomain antibody (5 μg/mL, R&D Systems, AB946) at 4°C overnight and then a secondary antibody, Alexa Fluor 594 donkey anti-goat (1:500, Life Technologies, Thermo Fisher Scientific, A11058) for 1 hour at room temperature.

Techniques: Control, Infection

Journal: Journal of Clinical Investigation

Article Title: Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis

doi: 10.1172/jci168450

Figure Lengend Snippet: Figure 4. Endothelial ADAM10 mediates platelet aggregation in a pathogen-specific manner. (A) Representative 2-photon images of control and VE-Cad ADAM10–/– mouse livers at 6–8 hours and (B) corresponding quantification of the total area of platelet accumulation within the vasculature of mouse livers after P. aeruginosa, S. pneumoniae, GBS, or C. albicans sepsis. Vasculature (red, Qdots655); platelets (green, GPIbβ). Scale bars: 20 μm. White arrows denote thrombi. Data represent 5–7 FOV per mouse in 5–6 male and female mice per group and indicate the mean ± SEM. Measurements for individual FOV within each mouse are displayed in Supplemental Figure 4, E–H. *P ≤ 0.05 and ***P ≤ 0.001, by nested t test.

Article Snippet: Vessels were then incubated in blocking buffer (PBS with 3% BSA, 1% fish gelatin, 0.5% Triton X-100) for 1 hour followed by a primary anti–mouse Adam10 ectodomain antibody (5 μg/mL, R&D Systems, AB946) at 4°C overnight and then a secondary antibody, Alexa Fluor 594 donkey anti-goat (1:500, Life Technologies, Thermo Fisher Scientific, A11058) for 1 hour at room temperature.

Techniques: Control

Journal: Journal of Clinical Investigation

Article Title: Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis

doi: 10.1172/jci168450

Figure Lengend Snippet: Figure 5. ADAM10 mediates platelet aggregation. (A) Representative 2-photon images of C57Bl/6 mice 6–8 hours after P. aeruginosa or S. pneumoniae sepsis, pretreated for 3 days with vehicle or the ADAM10 inhibitor GI254023X. Vasculature (red, Qdots655), platelets (green, GPIbβ). Scale bar: 20 μm. White arrows denote thrombi. (B) Quantification of the total area of platelet accumulation within the vasculature in mouse livers as treated in A. Data represent 5–7 FOV per mouse in 6–7 male and female mice per group and are presented as the mean ± SEM. Measurements for individual FOV within each mouse are displayed in Supplemental Figure 5, A and B. **P ≤ 0.01, by nested t test for each treatment group.

Article Snippet: Vessels were then incubated in blocking buffer (PBS with 3% BSA, 1% fish gelatin, 0.5% Triton X-100) for 1 hour followed by a primary anti–mouse Adam10 ectodomain antibody (5 μg/mL, R&D Systems, AB946) at 4°C overnight and then a secondary antibody, Alexa Fluor 594 donkey anti-goat (1:500, Life Technologies, Thermo Fisher Scientific, A11058) for 1 hour at room temperature.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A , comparison of TspanC8 co-immunoprecipitations with ADAM10 truncation constructs. Quantitation of the co-immunoprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from were compared. Values were normalized using Tspan14 data from . All data were relative to the co-immunoprecipitation of ADAM10DCS with Tspan14, which was arbitrarily set to 100. Data were log transformed and statistical analysis was performed using a one-way ANOVA with a Dunnett's multiple comparison test comparing ADAM10CS (#, p < 0.01) or ADAM10S (*, p < 0.01) to the ADAM10DCS for each tetraspanin. Error bars represent the standard error of the mean from three experiments. B , schematic of the potential differential modes of interaction of the TspanC8s with ADAM10. Bold regions of ADAM10 represent those required for a strong interaction with the corresponding TspanC8. Note that Tspan15 has 3 N -linked glycosylation sites and Tspan17 has 2, whereas Tspan5, 10, 14, and 33 have 3, 0, 1, and 2, respectively; for the latter, Tspan14 is depicted as an example.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Comparison, Construct, Quantitation Assay, Immunoprecipitation, Transformation Assay, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The combined cysteine-rich ( C ) and stalk ( S ) region of ADAM10 without the disintegrin ( D ) is sufficient to interact with Tspan14. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of Myc-tagged human ADAM10, or pDisplay constructs containing ADAM10DCS or ADAM10CS, which also possessed Myc tags. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). B , data in panel A ( upper panel ) were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett's multiple comparison test against the mock. Tspan14 bound significantly to ADAM10DCS ( p < 0.0001) and ADAM10CS ( p < 0.0001). A diagrammatic representation of the ADAM10 constructs is shown below the graph. C , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of pDisplay ADAM10CS or ADAM10S. Cells were treated as in panel A. D , data in panel C were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett's multiple comparison test against the mock. Tspan14 bound significantly to ADAM10CS ( p < 0.0001) and ADAM10S ( p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Cotransfection, Construct, Lysis, Immunoprecipitation, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The TspanC8s bind differentially to the disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse TspanC8s or CD9, and co-transfected with the pDisplay vector containing HA-tagged human ADAM10DCS. Cell lysates were produced in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). B , data from panel A ( upper panel ) were quantitated and presented as the amount of immunoprecipitated ADAM10DCS relative to the Tspan14 immunoprecipitation, which was arbitrarily set to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the CD9 control. All TspanC8s bound significantly to ADAM10DCS ( p < 0.001). Error bars represent the standard error of the mean from three experiments. C and D , these experiments were carried out as described for panels A and B except using HA-tagged human ADAM10CS. All TspanC8s bound significantly to ADAM10DCS ( p < 0.0001). E and F , these experiments were carried out as for panels A and B except using HA-tagged human ADAM10S (****, p < 0.0001; **, p < 0.01; *, p < 0.05).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Plasmid Preparation, Produced, Lysis, Immunoprecipitation, Transformation Assay, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Endogenous ADAM10 and Tspan14 interact in platelets and primary endothelial cells. A , HEK-293T cells were mock transfected (−) or transfected with a FLAG-tagged human Tspan14 expression construct (+). The cells were lysed in 1% Triton X-100 lysis buffer and subjected to anti-Tspan14 ( top panel ) and anti-FLAG ( lower panel ) Western blotting. The Tspan14 antibody was raised in goat against a C-terminal cytoplasmic peptide, in collaboration with Everest Biotech. B , washed human platelets; C , washed mouse platelets and D , human umbilical vein endothelial cells were lysed in 1% digitonin lysis buffer, and proteins were immunoprecipitated with an antibody against ADAM10 or an isotype-matched control. Precipitates were then run on non-reducing gels, Western blotted, and probed with Tspan14 ( top panels ), ADAM10 ( middle panels ), and CD9 ( lower panels ) antibodies. Arrows indicate the positions of the predominant mature form of ADAM10 (A10) and the signal from the immunoprecipitating antibodies (IgG).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Expressing, Construct, Lysis, Western Blot, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A , schematic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable (var) region of CD9 ( black ) and Tspan14 ( gray ) were interchanged; the N -linked glycosylation site of Tspan14 is indicated by a filled oval. B , HEK-293T cells were mock transfected (−) or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (+). Cell lysates were produced using 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). Data are representative of three independent experiments. C , quantitation of immunoprecipitated ADAM10. Data in panel B ( upper panel ) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock (****, p < 0.0001). Error bars represent standard error of the mean from three experiments. D , data in panel B ( middle panel ) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (***, p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Glycoproteomics, Transfection, Expressing, Construct, Produced, Lysis, Immunoprecipitation, Quantitation Assay, Imaging, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A , HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see A ) and GFP to identify transfected cells. Cells were stained with an APC-conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B , average geometric mean fluorescent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett's multiple comparison test, compared with the CD9 control (***, p < 0.001; **, p < 0.01). Error bars represent standard error of the mean from three experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Flow Cytometry, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: All Tspan14-CD9 chimeras partially co-localize with ADAM10 and so have access to the metalloprotease. HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see A ) and HA-tagged mouse ADAM10. Cells were fixed and stained with an anti-HA antibody ( green ) and an anti-FLAG antibody ( red ). Confocal microscopy images are representative of three independent experiments and at least 15 fields of view.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The region of ADAM10 comprising the disintegrin domain ( D ), the cysteine-rich ( C ), and stalk ( S ) regions mediates the interaction with Tspan14. A , schematic of ADAM10 and ADAM17 chimeras. The extracellular disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10 ( black ) and ADAM17 ( gray ) were interchanged together ( DCS ) or individually. B , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse Tspan14 (+) in addition to either HA-tagged mouse ADAM10, ADAM17, ADAM17 10DCS, or ADAM10 17DCS. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-HA tag antibody ( middle panel ). The blots are representative of three independent experiments. C , HEK-293T cells were co-transfected with (+) or without (−) FLAG-tagged mouse Tspan14 and either HA-mouse ADAM10, ADAM17, ADAM17 10DCS, ADAM17 10D, ADAM17 10C, or ADAM17 10S. Cells were treated as in B. D , data from panels B and C were quantitated and presented as the relative amount of each ADAM10/17 construct immunoprecipitated with Tspan14, having arbitrarily set wild-type ADAM10 to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test, compared with the ADAM17 control (*, p < 0.05). Error bars represent standard errors of the mean from 3–6 experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Lysis, Immunoprecipitation, Construct, Transformation Assay, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: All TspanC8s interact with the region of ADAM10 comprising the disintegrin ( D ), cysteine-rich domain ( C ), and stalk ( S ). A , HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or negative control (−). Lysates were extracted in 1% digitonin lysis buffer and proteins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-HA tag antibody ( middle panel ). B , data in panel A ( upper panel ) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Data are shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock. All TspanC8s bound significantly to ADAM17 10DCS ( p < 0.0001). Error bars represent standard error of the mean from three experiments. C , ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbitrarily set at 100. Error bars represent standard error of the mean from three experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Expressing, Construct, Negative Control, Lysis, Immunoprecipitation, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A , HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody ( green ), an anti-FLAG antibody ( red ) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B , HeLa cells were transfected and stained as in panel A except an anti-calnexin antibody was used instead of WGA to define the limits of the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10 or HA-ADAM10 17DCS signal localized in the ER. Data are representative of three independent experiments and at least 15 fields of view. A two-way ANOVA statistical analysis was performed with a Bonferroni's multiple comparisons test ( ns , non-significant, ****, p < 0.0001). C , HEK-293T cells were mock transfected (−), or transfected with HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HA antibody. Immunoprecipitates were stained with neutravidin ( top panel ) or an anti-HA antibody ( bottom panel ). Whole cell lysates were stained with an anti-HA antibody ( middle panel ).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Clinical Proteomics, Membrane, Confocal Microscopy, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Differential effects of TspanC8s on ADAM10 substrate cleavage: Tspan15 promotes cleavage of N-cadherin and Tspan14 reduces cleavage of GPVI. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse TspanC8s. The cells were lysed in 1% Triton X-100 lysis buffer and subjected to Western blotting with an antibody to the C-terminal cytoplasmic tail of N-cadherin ( upper panel ) or with an antibody to the FLAG epitope ( lower panel ). B , data from A ( upper panel ) were quantitated and the lower, cleaved band given as a percentage of the total ( upper and lower band combined). Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock control. Error bars represent the standard error of the mean from three experiments (*, p < 0.05). C , HEK-293T cells were co-transfected with GPVI and FcRγ and one of each of the FLAG-tagged mouse TspanC8s or without a tetraspanin (−) or with the addition of the ADAM10 inhibitor GI254023X at 10 μ m . Cells were treated as in panel A , except lysates were subjected to an anti-GFP antibody ( upper panel ) instead of an anti-N-cadherin antibody. D , data from panel C ( upper panel ) were quantitated as described in panel A (***, p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Lysis, Western Blot, FLAG-tag, Transformation Assay, Comparison, Control

Journal: Science Advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: ( A ) Immunofluorescence staining for MMP substrates, VCAM-1, NrCAM, agrin, NOTCH3, together with GFAP to mark astrocytes and DAPI; boxed areas are shown to the right at higher magnifications. Scale bars, 100 μm. ( B ) Silver-stained gels showing cleavage products of gelatinase substrates after overnight incubation without (0) or with 1:10 or 1:100 ratios of MMP-9:substrate or ADAM10:substrate. Arrows mark the positions of ADAM10 in samples. Asterisks mark specific cleavage products. Data are representative of two to three experiments.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl 2 , 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Immunofluorescence, Staining, Incubation