Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 1 | Genome-wide screen identifies PACS-2 as an ADAM17 regulator. (a) Cellular model system used for the genome-wide siRNA screen. A total of 21,121 individual genes were knocked down using siRNAs and the effects on ADAM17-mediated shedding of AP-HB-EGF were measured by quantifying AP cell-surface staining after PMA stimulation. (b, c) siRNA-transfected AP-HB-EGF HT1080 (b) or AP-HB-EGF HeLa (c) cells were PMA-stimulated, and the cell medium was analysed for AP activity. The fold change in AP-HB-EGF release was calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value, and finally calculating the average of all individual experiments. Data in b were compiled from six individual experiments and in c from three individual experiments, each performed in triplicate. (d) AP-HB-EGF MCF-7 cells were siRNA- transfected and stimulated with PMA (left-hand panel) or ionomycin (right-hand panel). Conditioned medium was analysed for AP activity as in b and c. Data were compiled from four individual experiments, each performed in triplicate. In all cases, knockdown was confirmed by western blot. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Genome Wide, Staining, Transfection, Activity Assay, Negative Control, Knockdown, Western Blot

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 2 | PACS-2 regulates ADAM17-mediated shedding. (a) AP-HB-EGF was transiently expressed in control and Pacs2 / MEFs. Cells were PMA-stimulated, and the cell medium was analysed for AP activity. To overcome differences in transfection efficiency, the fraction of shed AP-HB-EGF was calculated as AP-HB-EGF released to the medium divided by the total amount of AP-HB-EGF in the medium and cell lysate. The fold change in AP-HB-EGF release was then calculated by setting the unstimulated negative control for each experiment to 1, normalizing the other raw data to this value and finally calculating the average of all individual experiments. The PMA-stimulated fold increase in AP-HB-EGF shedding was depicted for each cell line. Data were compiled from four individual experiments, each performed in triplicate. (b) AP-HB-EGF was expressed in Pacs2 / MEFs together with green fluorescent protein (GFP) or PACS-2-HA and shedding analysed as in a. Data were compiled from three individual experiments, each performed in triplicate. (c–e) siRNA-transfected MDA-MB-231 cells were stimulated with PMA (c,d) or TNF-a (e). The medium was analysed by ELISA for shed HB-EGF (c) or TGF-a (d,e), shown as concentrations in pg ml 1. In all cases, data were compiled from three individual experiments, each performed in triplicate. (f) Representative western blots showing knockdown in cells used for ELISA. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Control, Activity Assay, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 3 | PACS-2 regulates cell-surface availability of mature ADAM17. (a,b) MDA-MB-231 cells were siRNA-transfected and PMA-stimulated. Cells were surface biotinylated and analysed by western blot. ADAM17 levels were normalized to input actin and fold changes were calculated by setting the unstimulated negative control for each experiment to 1, then normalizing the other raw data to this value and finally calculating the average of all individual experiments. Cell-surface levels of mature ADAM17 (a) and total cellular levels of pro and mature ADAM17 (b) are shown with data compiled from four individual experiments. (c,d) Unstimulated cell-surface levels of mature ADAM17 (c) and total cellular levels of pro and mature ADAM17 (d) in control and Pacs2 / MEFs were analysed as in a and b. Data were compiled from four individual experiments in c and three individual experiments in d. (e) ADAM17 was immunoprecipitated from unstimulated control and Pacs2 / MEFs and its activity assessed using an ADAM17 quenched fluorescence peptide substrate. The graph shows the average gradient (fluorescence units/time) of the linear regressions describing enzymatic activity. The data were compiled from three individual experiments, each performed in triplicate. (f) Lysates from control and Pacs2 / MEFs were treated with EndoH or PNGase F and the effects on ADAM17 examined by western blot. The images shown are derived from the same blot but different exposures (indicated by separate panels). The blot shown is representative of three individual experiments. On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by analysis of variance or unpaired two-tailed Student’s t-test, as appropriate. **Po0.01, ***Po0.001, ****Po0.0001.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Transfection, Western Blot, Negative Control, Control, Immunoprecipitation, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 4 | PACS-2 interacts with ADAM17 in endocytic compartments. (a,b) ADAM17 (a) or PACS-2 (b) was immunoprecipitated (IP) from unstimulated and PMA-stimulated MDA-MB-231 cells, and co-immunoprecipitation (co-IP) of PACS-2 (a) or ADAM17 (b) detected by western blot. The blots shown are representative of three individual experiments and # denotes a nonspecific band. (c) The effect of PMA stimulation on PACS-2/ADAM17 co-immunoprecipitation in a and b was quantified. The amount of co-immunoprecipitated PACS-2 (a) or mature ADAM17 (b) was normalized to the amount of immunoprecipitated mature ADAM17 (a) or PACS-2 (b). The unstimulated negative control for each experiment was then set to 1, the other raw data were normalized to this value and finally the average of all individual experiments was calculated. Data were analysed by unpaired two-tailed Student’s t-test. (d) MDA-MB-231 cells were transfected with siRNA and subjected to PLA with or without PMA stimulation. The experiment was performed 10 times in duplicate. Two experiments included the knockdown controls. Scale bar, 7 mm. (e) Before PLA, MDA-MB-231 cells were left unstimulated or treated with PMA and subsequently allowed to internalize fluorescent transferrin for 5 min. Scale bar, 14 mm. Fluorescence intensities were quantified along the lines on the enlarged images and depicted on the graphs. The experiment was performed four times in duplicate.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Two Tailed Test, Transfection, Knockdown, Fluorescence

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 5 | PACS-2 diverts endocytosed ADAM17 away from degradative pathways. (a) MDA-MB-231 cells were surface labelled using cleavable biotin. Labelled cell-surface proteins were allowed to internalize for 30 min and remaining biotinylated proteins on the cell surface were removed (Intern.). Internalized proteins were then allowed to recycle for 30 min, after which recycled protein was stripped from the cell surface (Recyc.). The amount of cell-surface ADAM17 was first normalized to input actin. The percentage internalization was calculated relative to total cell-surface ADAM17 levels (Total). Recycling was determined by first subtracting the amount of biotinylated protein left after recycling from the amount of internalized protein, and then calculated as a percentage of internalized protein. To verify proper stripping of cell-surface proteins, a dish kept at 4 C was processed in parallel (Strip). Data were compiled from five individual experiments. (b) MDA-MB-231 cells were surface labelled using non-cleavable biotin, lysed at time zero, or incubated for 4 h at 37 C to allow internalization and degradation of surface proteins. Percentage degradation was calculated by normalizing the amount of cell-surface ADAM17 to input actin, and dividing the amount remaining after 4 h with the amount present at time zero. Data were compiled from three individual experiments. The images shown are derived from the same blot and same exposure, but have been cropped for clarity (indicated by a separation line). On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by unpaired two-tailed Student’s t-test. *Po0.05.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Stripping Membranes, Incubation, Derivative Assay, Two Tailed Test

Journal: Nature communications

Article Title: The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17.

doi: 10.1038/ncomms8518

Figure Lengend Snippet: Figure 8 | A model of PACS-2 regulation of ADAM17 and EGFR activity. The ADAM17 proform mainly resides in the secretory apparatus, and during its transit to the cell surface, furin-mediated cleavage generates the mature (active) form. We propose that PACS-2 controls ADAM17 cell-surface availability by diverting endocytosed ADAM17 away from degradative pathways and towards sustained ErbB ligand shedding and EGFR activation. PACS-2 is therefore an important regulator of auto, para and juxtacrine ErbB signalling in vivo.

Article Snippet: Primary antibodies were rabbit PACS-2 antibody (Proteintech 19508-1-AP) and mouse ADAM17 antibody (R&D Systems MAB9301).

Techniques: Activity Assay, Activation Assay, In Vivo

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: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 2. Downregulation of the mature ADAM17 form by PMA. A-C) A549 cells were stimulated with PMA (200 ng/ml) in serum free medium for the indicated time periods. Subsequently, cells were lysed for western blotting (A, B) or subjected to biotinylation of surface proteins prior to cell lysis and subsequent precipitation of surface proteins by streptavidin sepharose (C). Cell lysates and precipitates were then probed with antibodies against the C-terminus of ADAM10 (B) and ADAM17 (A, C), respectively. The signal intensity for the pro-form and the mature form of the proteases was determined by densitometry, expressed in relation to the unstimulated controls for each time point and summarized as mean and SD from three independent experiments. Differences to the control were analyzed by two tailed one sample t-test and statistically significant differences indicated as asterisks (p b 0.05).

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Western Blot, Lysis, Control, Two Tailed Test

Journal: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 4. Role of dynamin in PMA-induced ADAM17 surface regulation. A-C) A549 cells were pretreated for 30 min in serum free medium with the dynamin inhibitor dynasore (100 μM) and subsequently PMA (200 ng/ml) was added. DMSO (0.1%) served as control. After 2 h, cells were analyzed for surface expression of ADAM10 and ADAM17 (A, B) or for mRNA expression (C, D) of the proteases. E, F) A549 cells were transfected with siRNA directed against dynamin or irrelevant control siRNA and then stimulated with PMA (200 ng/ml) for 2 h or left unstimulated. Successful downregulation was monitored by Western blotting for dynamin (E) and the effect on ADAM17 surface regulation was determined by flow cytometry (F). Results were calculated in relation to the control and represent means and SD from three independent experiments. Differences due to dynamin targeting were analyzed by t-test and indicated by asterisks when significant (p b 0.05).

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Control, Expressing, Transfection, Western Blot, Cytometry

Journal: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 5. Exosomal release of mature ADAM17. A) A549 cells were stimulated with PMA (200 ng/ml) or were left unstimulated (0.1% DMSO) in serum free medium for 24 h. Subsequently, cells were lysed and the cell supernatant was subjected to differential centrifugation at the indicated forces. Before the final centrifugation step the supernatant was cleared by passing through 0.2 μm filters. Cell lysates, unfractionated supernatants and each sediment obtained by centrifugation were analyzed for ADAM10 and ADAM17 immunoreactivity by Western blotting. β-actin served as a loading control for cell lysates. B) A549 cells were stimulated with PMA (200 ng/ml) or were left unstimulated (0.1% DMSO) for 4, 6 and 24 h and subjected to differential centrifugation as in A. The extracellular microvesicle fraction obtained by centrifugation at 100,000 x g was analyzed by Western blotting for ADAM10, ADAM17 and exosomal markers Hsp70 and flotillin-1. C) Cells were stimulated and supernatants were subjected to differential centrifugation as in A. Cell lysates and the extracellular microvesicle fraction were analyzed by Western blotting for ADAM10, ADAM17 and exosomal markers Hsp70, flotillin-1, CD9. D) Microvesicles were prepared from supernatants of PMA-stimulated A549 cells as in B and subsequently further fractionated by sucrose density gradient centrifugation. The ADAM10 and ADAM17 positive fractions were identified as exosomal fractions containing the exosomal markers. The data shown are representative for at least three independent experiments.

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Centrifugation, Western Blot, Control, Gradient Centrifugation

Journal: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 6. Exosomal ADAM17 release by LPS-stimulated monocytic, epithelial and primary endothelial cells. A) A549 cells and THP-1 cells were stimulated with LPS (50 μg/ml) and LBP (0.1 μg/ml) or left unstimulated for 24 h. Subsequently, exososmal release of ADAM10 and ADAM17 was investigated by Western blotting. Hsp70 and CD9 served as exosomal markers. B) HUVECs were stimulated with LPS (5 μg/ml), PMA (200 ng/ml) or left unstimulated for 24 h. Subsequently, exosomal release of ADAM10 and ADAM17 was investigated. Hsp70, flotillin-1 and CD9 served as exosomal markers. In parallel, cell lysates were analyzed for the presence of mature ADAM10 and ADAM17. GAPDH served as loading control. The data are shown as representative of three (A) or two (B) independent experiments.

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Western Blot, Control

Journal: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 7. Exosomal release of surface expressed ADAM17. A) Exosomes were prepared from supernatants of PMA-stimulated A549 cells and either resuspended in PBS or lysis buffer. Intact vesicles or lysed membranes, respectively, were bound to aldehyde/sulphate latex beads and probed with antibodies against the N-terminus of ADAM10 or ADAM17 or with an antibody against the C-terminus of ADAM17. Bound antibodies were detected with fluorochrome-coupled secondary antibodies and the fluorescence signal was analyzed by flow cytometry. B) A549 cells were transduced with lentivirus encoding ADAM17 shRNA or control shRNA. Cells were labelled with APC-coupled antibody against the N-terminus of ADAM17 or APC- coupled isotype control for 30 min. After 24 h stimulation with PMA (200 ng/ml), exosomes were prepared from the cell culture supernatants, precipitated with aldehyde/sulphate latex beads and investigated for bound fluorescence by flow cytometry. Beads without exosomes served as a control. C) A549 cells were transduced with shRNA against iRHOM2 (549 and 550) or control shRNA. Subsequently cells were stimulated with PMA and studied for release of ADAM10 and ADAM17 in exosomes by Western blotting. GAPDH served as a loading control. Hsp70 and flotillin-1 served as exosomal markers. The data are shown as representative of three independent experiments.

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Lysis, Cytometry, Transduction, shRNA, Control, Cell Culture, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Stimulated release and functional activity of surface expressed metalloproteinase ADAM17 in exosomes.

doi: 10.1016/j.bbamcr.2016.09.002

Figure Lengend Snippet: Fig. 8. Requirements of exosomal ADAM17 release. A) HEK293 cells were transfected to express murine ADAM17 or a C-terminally truncated murine ADAM17 variant lacking the cytoplasmic region and stimulated with PMA (200 ng/ml) or left unstimulated. After 24 h, exosomes were prepared from cell culture supernatants and analyzed for ADAM17 immunoreactivity. Hsp70 served as exosomal loading control. In a different set of experiments cells were lysed for Western blotting or subjected to biotinylation of surface proteins prior to cell lysis and subsequent precipitation of surface proteins by streptavidin sepharose. Cell lysates and precipitates were then probed with antibodies against the N-terminus of ADAM17. GAPDH served as cytosolic loading control. B) A549 cells were left unstimulated or stimulated with PMA (200 ng/ml) in the presence or absence of BAPTA-AM (30 μM). After 24 h of incubation, released exosomes were investigated for ADAM17, ADAM10, flotillin-1 and Hsp70 immunoreactivity. C) Surface expression of ADAM17 was investigated by flow cytometry on cells stimulated with PMA in the presence or absence of BAPTA-AM. Data in A-D are shown as representative for at least three independent experiments. Data in E were calculated in relation to the control and represent means and SD from three independent experiments. Differences due to BAPTA treatment were statistically analyzed by t-test and indicated by asterisks when significant (p b 0.05).

Article Snippet: For flow cytometry unconjugated and PE-coupled mouse monoclonal antibodies (mab) to ADAM17 ectodomain (# 111,633) and mab to ADAM10 ectodomain (# 163,003), mouse IgG2b, IgG1 and allophycocyanin (APC)-coupled IgG1 isotype controls, and normal rabbit IgG were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Variant Assay, Cell Culture, Control, Western Blot, Lysis, Incubation, Expressing, Cytometry

Journal: Journal of Biological Chemistry

Article Title: Acidic Mammalian Chitinase Is Secreted via an ADAM17/Epidermal Growth Factor Receptor-dependent Pathway and Stimulates Chemokine Production by Pulmonary Epithelial Cells

doi: 10.1074/jbc.m805574200

Figure Lengend Snippet: FIGURE 3. Th2 airway inflammation increases EGFR and ADAM17 expression in lung epithelial cells. a, immunoblotting of EGFR expression in whole lung cell lysates in PBS-sensitized and challenged (PBS) and OVA-sensitized and challenged mice (OVA) (top). WT and IL-13-overexpressing transgenic mice (IL-13 tg) are seen in the lower panel. b, EGFR expression in lung epithelial cells using immunohistochemistry in WT and IL-13-overexpressing transgenic mice. In the upper panel an isotype control IgG antibody was used instead of the specific anti-EGFR antibody. c, EGFR surface expression on CD45CD31cytokeratin cells in WT and IL-13-overexpressing transgenic mice, PBS-sensitized and -challenged mice, and OVA-sensitized and -chal- lenged mice. Bars, means S.D. (*, p 0.05; Student’s t test). d, representative histogram of EGFR surface expression on CD45CD31cytokeratin cells. The left panel shows EGFR expression in lungs from IL-13- overexpressing transgenic mice, and the right panel shows expression in lungs from OVA-sensitized and -challenged (OVA) mice. Green line, isotype (ISO) control; filled blue area, EGFR expression on CD45CD31cytokeratin cells of WT mice; red line, EGFR expression on CD45CD31cytokeratin cells from IL-13-overexpressing transgenic mice. e, AMCase (Alexa-488, green dye) and EGFR (Cy3, red dye) were localized in tissues from IL-13-overexpressing transgenic mice. Confocal microscopy was performed with a Zeiss LSM 510 META system. Co-localization of AMCase and EGFR after merging was observed as a yellow color with confocal microscopy. f, ADAM17 surface expression was evaluated on CD45CD31cytokeratin lung epithelial cells from WT and IL-13-overexpressing transgenic (left) mice, PBS-sensitized and challenged mice, and OVA-sensitized and challenged mice (right) (p 0.05; Student’s t test). g, representative histogram of ADAM17 expression on CD45CD31cytokeratin cells. Green line, isotype control. The filled blue area and the red line illustrate the ADAM17 expression on CD45CD31cytokeratin cells from WT and IL-13-overexpress- ing transgenic mice, respectively. MFI, mean fluorescence intensity.

Article Snippet: Anti-human ADAM17 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and employed as reported previously (20).

Techniques: Expressing, Western Blot, Transgenic Assay, Immunohistochemistry, Control, Confocal Microscopy, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Acidic Mammalian Chitinase Is Secreted via an ADAM17/Epidermal Growth Factor Receptor-dependent Pathway and Stimulates Chemokine Production by Pulmonary Epithelial Cells

doi: 10.1074/jbc.m805574200

Figure Lengend Snippet: FIGURE 4. AMCase, EGFR, and ADAM17 colocalize in lung epithelial cells. AMCase, EGFR, and ADAM17 expression were quantified within CD45CD31cytokeratin cells in IL-13-overexpressing transgenic mice. a and b show AMCase coexpression with EGFR (a) and ADAM17 (b), and c shows coexpression of EGFR with ADAM17. The respective isotype controls are depicted.

Article Snippet: Anti-human ADAM17 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and employed as reported previously (20).

Techniques: Expressing, Transgenic Assay

Journal: Journal of Biological Chemistry

Article Title: Acidic Mammalian Chitinase Is Secreted via an ADAM17/Epidermal Growth Factor Receptor-dependent Pathway and Stimulates Chemokine Production by Pulmonary Epithelial Cells

doi: 10.1074/jbc.m805574200

Figure Lengend Snippet: FIGURE 7. ADAM17 and Ras modulate AMCase secretion. a, A549 cells were transfected with empty vector (AMCase ) or AMCase (AMCase ) and were treated with the EGFR inhibitors PD153035 (1 M) or AG1478 (1 M), the ADAM17 inhibitors TAPI-1 (25 M) and TAPI-2 (25 M), the ERK inhibitor PD98059 (10 M), or the Ras/Raf inhibitor manumycin A (1 M). Supernatant chitinase activity was measured after 48 h of cell culture (*, p 0.05 chitinase activity in supernatants of untreated versus inhibitor-treated AMCase-transfected A549 cells; Mann-Whitney U test). Supernatant AMCase was evaluated by Western blotting. b, A549 cells were transfected with AMCase and were treated with manumycin A (MA) (at the indicated doses; 1 nM to 10 M), PD98059 (1 nM to 10 M), or PBS. Supernatant chitinase activity (top) and IL-6 levels (bottom) were measured after 48 h of cell culture (*, p 0.05, Mann-Whitney U test; #, p 0.05, Mann-Whitney U test versus untreated).

Article Snippet: Anti-human ADAM17 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and employed as reported previously (20).

Techniques: Transfection, Plasmid Preparation, Activity Assay, Cell Culture, MANN-WHITNEY, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease.

doi: 10.1038/labinvest.3700465

Figure Lengend Snippet: Figure 1 Normal colon. (a) A strong expression of ADAM15 by colonic epithelial cells and endothelial cells of the mucosa (M) and submucosa (SM). ADAM15 is weakly expressed by smooth muscle cells of the muscularis mucosae (MM); (b) ADAM15-positive staining at the basolateral membrane of normal epithelial cells; (c) ADAM15 immunostaining of the muscularis propria (MP); (d) negative control. Scale bars: 100 mm.

Article Snippet: After blocking, membranes were incubated with a goat polyclonal antibody against human ADAM15 cytoplasmic tail (dilution 1:100; R&D systems) or with a mouse monoclonal antibody against human b-actin (dilution 1:10 000; Sigma), and then with a horseradish-peroxidase conjugated, rabbit anti-goat antibody (dilution 1:20 000; Biodesign Int., Kennebunk, ME, USA) or goat antimouse antibody (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Membrane, Immunostaining, Negative Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease.

doi: 10.1038/labinvest.3700465

Figure Lengend Snippet: Figure 2 Normal colon. (a, b) Double immunostaining of ADAM15 (green), and a-SMA (red, a) or collagen IV (red, b), was performed on paraformaldehyde-fixed frozen sections and examined by confocal microscopy, as described in Materials and methods. Nuclei appear in blue. ADAM15 is strongly expressed by epithelial cells, and by pericryptic myofibroblasts, which coexpress a-SMA and collagen IV. (c, d) Double immunostaining of a5b1 (green, c) or avb3 (green, d) and a-SMA (red) was performed on acetone-fixed frozen sections and examined by confocal microscopy, as described in Materials and methods. Nuclei appear in blue. Pericryptic myofibroblasts, scoring positive for a-SMA, strongly express a5b1 and avb3, as well as muscularis mucosae myocytes. Scale bars: 20 mm.

Article Snippet: After blocking, membranes were incubated with a goat polyclonal antibody against human ADAM15 cytoplasmic tail (dilution 1:100; R&D systems) or with a mouse monoclonal antibody against human b-actin (dilution 1:10 000; Sigma), and then with a horseradish-peroxidase conjugated, rabbit anti-goat antibody (dilution 1:20 000; Biodesign Int., Kennebunk, ME, USA) or goat antimouse antibody (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Double Immunostaining, Confocal Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease.

doi: 10.1038/labinvest.3700465

Figure Lengend Snippet: Figure 3 Inflammatory bowel disease. (a) Strong expression of a5b1-integrin by pericryptic myofibroblasts and polymorphonuclear cells (arrowheads) (original magnification 200); (b) Polymorphonuclear cell diapedesis through endothelial cells expressing ADAM15; (c and d) VE-cadherin expression is decreased at the membrane of endothelial cells (d), as compared with normal control (c). Scale bars: 50 mm.

Article Snippet: After blocking, membranes were incubated with a goat polyclonal antibody against human ADAM15 cytoplasmic tail (dilution 1:100; R&D systems) or with a mouse monoclonal antibody against human b-actin (dilution 1:10 000; Sigma), and then with a horseradish-peroxidase conjugated, rabbit anti-goat antibody (dilution 1:20 000; Biodesign Int., Kennebunk, ME, USA) or goat antimouse antibody (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Membrane, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: ADAM15 upregulation and interaction with multiple binding partners in inflammatory bowel disease.

doi: 10.1038/labinvest.3700465

Figure Lengend Snippet: Figure 4 Inflammatory bowel disease: regenerative changes, (a) immunohistochemistry: Strong expression of ADAM15 in regen- erative cryptic epithelial cells with mucus depletion (arrowheads) (Scale bar: 50 mm); (b) immunoblot: A representative immunoblot shows that the active form of ADAM15 protein (90 kDa) is expressed in normal colon, as well as in ulcerative colitis and in Crohn’s disease. Immunoblot analysis was performed as described in Materials and methods from cell lysates of human colon (lane 1), of ulcerative colitis (lane 2) and of Crohn’s disease (lane 3) (50 mg protein loaded) using polyclonal goat anti- ADAM15 ectodomain or anti-b actin antibodies. Numbers on the right are the molecular size of standards in thousands.

Article Snippet: After blocking, membranes were incubated with a goat polyclonal antibody against human ADAM15 cytoplasmic tail (dilution 1:100; R&D systems) or with a mouse monoclonal antibody against human b-actin (dilution 1:10 000; Sigma), and then with a horseradish-peroxidase conjugated, rabbit anti-goat antibody (dilution 1:20 000; Biodesign Int., Kennebunk, ME, USA) or goat antimouse antibody (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Expressing, Western Blot

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Representative Western blots showing membrane levels of PrP C as revealed by surface biotinylation (I; on the left) and total PrP C levels in lysates (I; on the right) as well as ADAM10 surface expression (II) of neuronally differentiated neural stem cells (NSCs) from Nestin A10 KO and littermate control mice and after genetic reintroduction of Adam10 (A10 KO + ADAM10) or nucleofection with control vector (A10 KO + Vector) into NSCs of Nestin A10 KO mice. Flotillin served as loading control. (III) Quantification of densitometric analysis of PrP C membrane levels of experimental groups mentioned above (n = 9 independent samples for controls [set to 1]; n = 9 for Nestin A10 KO; n = 8 for Nestin A10 KO + ADAM10; n = 5 for Nestin A10 KO + Vector; significance: **p = 0.0054; ## p = 0.0014; *p = 0.0336 ; # p = 0.0212). Error bars indicate SEM. ( B ) Representative immunofluorescent PrP C (green) surface staining of neuronally differentiated NSCs derived from Nestin A10 KO (without [second row] or with genetic reintroduction of ADAM10 [third row] or vector only [fourth row]) and littermate control mice (first row), respectively. Tubulin (red) was stained after permeabilization of cells to confirm neuronal differentiation of NSCs. DAPI (blue) marks nuclei. ( C ) Representative immunostaining of PrP C (green) and ADAM10 (red) in permeabilized (upper two rows) and non-permeabilized (lower three rows) murine embryonic fibroblasts (MEFs) derived from mice with a complete knockout of ADAM10 (ADAM10 KO) or wild-type mice (control). Higher resolution of white boxes is shown in the bottom row and reveals colocalization of PrP C and ADAM10 at the plasma membrane of wild-type control MEFs. Scale bars in B and C represent 10 µm. ( D ) Western blot analysis of cell-associated PrP C levels in ADAM10 knockout (A10 KO) and wild-type (wt) MEF lysates (left part: actin served as loading control). Levels of shed PrP C were assessed in cell culture media supernatants of ADAM10 knockout and wild-type MEFs by filter column concentration (conc. media) and immunoprecipitation (IP) with a PrP C -specific antibody respectively (right part). ( E ) Levels of cell-associated (neuronal lysates) and shed PrP C (IP of media supernatants) in primary neuronal cultures of prion protein knockout ( Prnp 0/0 ), wild-type (wt; C57BL/6), prion protein overexpressing ( tg a 20 ), and Nestin A10 KO mice at embryonic day 14. IgG-HC and IgG-LC mark signals for heavy and light chain of the capturing antibody POM2. DOI: http://dx.doi.org/10.7554/eLife.04260.003

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Membrane, Expressing, Control, Plasmid Preparation, Staining, Derivative Assay, Immunostaining, Knock-Out, Clinical Proteomics, Cell Culture, Concentration Assay, Immunoprecipitation

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Western blot analysis of mature ADAM10 (mADAM10) and PrP C expression in different brain homogenates from the cortex (left) and cerebellum (right) of A10 cKO and control mice at postnatal day (P) 19. Actin served as a loading control. ( B ) Immunohistochemical detection of PrP C in forebrain of P19 mice of both genotypes. Overview (top) and magnifications showing cortex (Cx) and hippocampal CA2 and CA3 regions. Overview and details of cerebellum (Cb) are shown below. Scale bars represent 100 µm (insets for Cb: 50 µm). ( C ) Quantitative RT-PCR analysis of PrP C mRNA levels in A10 cKO mice and controls at P19 (n = 5 for each genotype). GAPDH served as a control for normalization. Error bars indicate SD. ( D ) Adult age-matched A10 cKO and wild-type littermates had a comparable body size at 35 weeks of age. ( E ) Representative immunohistochemical staining of PrP C in cortex (Cx) and hippocampal CA1 region of adult (35 weeks) A10 cO and control mice. Prion protein knockout ( Prnp 0/0 ) and overexpressing mice ( tg a 20 ) served as negative and positive controls, respectively (scale bar: 100 µm). DOI: http://dx.doi.org/10.7554/eLife.04260.004

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Expressing, Control, Immunohistochemical staining, Quantitative RT-PCR, Staining, Knock-Out

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Scheme of a mouse brain combining a qualitative representation of the Camk2a driven ADAM10 knockout strategy and information on the sampling of specimen. The site of intracerebral inoculation of mice with RML prions is indicated by the red encircled dot. Samples of frontal brain (dotted box) were taken for biochemical analysis and determination of infectivity titers (bioassay). The rest of the brain was formalin-fixed and embedded in paraffin. Coronal sections were prepared from different layers (dashed lines) with varying distance to the site of prion inoculation (as indicated by blue arrows) and assessed by immunohistochemical analysis. ( B ) Qualitative comparison of mouse genotypes (A10 cKO, controls, tg a 20 ) with regard to PrP C or PrP Sc levels, prion-associated neuropathology (including spongiosis, astrocytosis, and microglia activation) and prion infectivity titers according to brain region and time point. Reference to corresponding figures showing original data is provided. Cb = Cerebellum; Cx = Cortex; Hc = Hippocampus; Stri = Striatum; Tha = Thalamus; n.a. = not assessed; Qualities: +++ = high/strong; ++ = medium/moderate; + = low/basal; (+) = very low/weak; o = none. DOI: http://dx.doi.org/10.7554/eLife.04260.007

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Knock-Out, Sampling, Infection, Bioassay, Immunohistochemical staining, Comparison, Activation Assay

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Representative histological analysis in the thalamic brain region of terminally prion-diseased ADAM10 cKO and control mice including H&E staining and immunohistochemical detection of PrP Sc , GFAP (for detection of astrocytes), and Iba-1 (for detection of microglia). Scale bar: 100 µm. DOI: http://dx.doi.org/10.7554/eLife.04260.011

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Control, Staining, Immunohistochemical staining

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Overviews showing vacuolization (asterisks) in forebrain samples of ADAM10 cKO ( A ) and control mice ( B ) at a terminal stage of prion disease. High abundance of clusters containing tubulovesicular structures (TVS; arrows in ( A ) and ( C )) was only found in terminal ADAM10 cKO brain (5 out of 13 square grids showed clusters of TVS whereas only 1 out of 15 square grids presented with TVS in terminal wild-type control mice). Scale bars represent 500 nm ( A , B ) or 200 nm ( C ). Inset in ( C ) shows magnification of TVS. DOI: http://dx.doi.org/10.7554/eLife.04260.012

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Assessment of total PrP (no proteinase K (PK)) and PrP Sc amounts (+PK; blot is shown with short and longer exposition) by parallel replica Western blot analysis in forebrain homogenates of age-matched A10 cKO mice and littermate controls (both at 95 days post inoculation (dpi); n = 3 for each genotype) as well as terminally diseased tg a 20 mice (at 65 dpi; n = 3). Actin was detected in the undigested homogenates (no PK) and served as loading control. Densitometric quantification of relative protein amounts from two technical replicates is shown on the right. ( B ) Morphological analysis of neuropathological lesions in forebrains (showing hippocampal and cortical brain regions) of A10 cKO, littermate controls, and tg a 20 mice at the aforementioned time points (scale bars: 200 µm in overviews and 100 µm in insets and for PrP Sc ). ( C ) Biochemical assessment of candidate toxic signaling pathways showing protein levels of total Fyn (t-Fyn), phosphorylated (Tyr416) Src (p-Src) as well as total (t-Erk1/2) and phosporylated (Thr202/Tyr204) Erk1/2 (p-Erk1/2). Actin served as a loading control and for normalization (# and § indicate use of the same actins as corresponding signaling proteins were detected on the same Western blot). Quantitative densitometric analysis of relative p-Src/t-Fyn (left) and p-Erk/t-Erk ratio (right) (n.s. = not significant). ( D ) Representative Western blot analysis (left) and quantification of three technical replicates (right) of calpain levels in aforementioned samples. Levels of ADAM10 are shown in ( C ) and ( D ) to confirm the ADAM10 status. Error bars indicate SEM; *p <0.05; **p <0.01, ***p <0.001 (p values of Student's t-test are given in the main text). DOI: http://dx.doi.org/10.7554/eLife.04260.013

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Control, Protein-Protein interactions

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ( A ) Representative Western blot analysis of calpain expression in non-prion infected ADAM10 cKO and control mice. In addition, premature (pADAM10), mature ADAM10 (mADAM10) and PrP C were detected. Quantification of calpain levels by densitometric analysis of ADAM10 cKO (n = 4) and control mice (n = 5) is shown below. Actin served as loading control. ( B ) Western blot analysis of spectrin (FL = full length) and spectrin breakdown products (SBDP) in prion-infected ADAM10 cKO and control mice at 95 days post inoculation (dpi; n = 3 per genotype). FL spectrin was reduced in ADAM10 cKO mice whereas calpain-dependent SBDP (marked by asterisk at 150/145 kDa) were not increased. ( C ) Western blot analysis of p35 and p25 levels in prion-infected samples mentioned in ( B ). A reduction in p35 levels was found in two out of three ADAM10 cKO mice. A band corresponding to p25 was not detectable. # indicates that blots used for calpain detection were re-probed with an antibody against p35/p25 and thus the same actin signals were used as loading controls. DOI: http://dx.doi.org/10.7554/eLife.04260.015

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Western Blot, Expressing, Infection, Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: Representative histological analysis of spongiotic vacuolization as revealed by H&E staining of the cerebellum and brain stem of terminally prion-diseased control and ADAM10 cKO mice. Upper row shows overview and lower two rows show higher magnifications of cerebellum and brain stem (scale bars represent 100 µm). DOI: http://dx.doi.org/10.7554/eLife.04260.018

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Staining, Control

Journal: eLife

Article Title: The sheddase ADAM10 is a potent modulator of prion disease

doi: 10.7554/eLife.04260

Figure Lengend Snippet: ADAM10 regulates PrP C levels at the plasma membrane and releases almost full length PrP into the extracellular space. Thereby it affects (i) neurotoxicity, (ii) PrP Sc formation, and (iii) spreading of prion pathology. (i) Lack of ADAM10 (as assessed here by use of A10 cKO mice) results in elevated PrP C membrane levels. Membrane levels of PrP C (as a receptor) likely determine PrP Sc -associated neurotoxicity (as indicated by sizes of thunderbolts and skulls) and thereby incubation times with shortest survival in tg a 20 mice and reduced incubation times in A10 cKO mice compared with wild-type littermates with longest survival (order reflected by grey triangles on the left). (ii) Shed PrP, which is most efficiently produced in tg a 20 and absent in A10 cKO mice, might block formation of PrP Sc . This is reflected by the different PrP Sc amounts found in our different experimental groups (A10 cKO > wild-type littermates > tg a 20 ). The combination of increased PrP C membrane levels and PrP Sc formation in A10 cKO mice might favor increased production of membrane pores (as indicated in the middle row on the left) and neurotoxic Ca 2+ influx with possible (‘?’) involvement of calpain. (iii) Finally, spread of prion-associated pathology within the brain also seems to be affected by the levels of ADAM10 expression since tg a 20 mice showed enhanced whereas A10 cKO mice showed reduced dissemination of neuropathological features (as indicated by size of arrowheads on the right). Key references supporting this model are given in the text. Based on this model, stimulation of ADAM10 might therefore offer a treatment option. With regard to incubation times, protective effects by reducing local membrane bound PrP C amounts and by producing a protective soluble fragment able to block PrP Sc formation seem to predominate the disadvantage of increased spread by production of anchorless prions. DOI: http://dx.doi.org/10.7554/eLife.04260.019

Article Snippet: For MEF cells, surface staining of PrP C and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively.

Techniques: Clinical Proteomics, Membrane, Incubation, Produced, Blocking Assay, Expressing

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Frozen lung Cohort: Demographic and clinical characteristics and ADAM15 gene expression levels

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Gene Expression

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Bronchoalveolar lavage (BAL) cohort: Demographic and clinical characteristics and ADAM15 levels

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Affinity Magnetic Separation

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Plasma cohort: Demographic and clinical characteristics and soluble ADAM15 levels

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Clinical Proteomics

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: A Disintegrin and A Metalloproteinase Domain 15 (ADAM15) expression is increased in lung samples and alveolar macrophages (AMs) from patients with chronic obstructive pulmonary disease (COPD) versus controls. In a , ADAM15 steady state mRNA levels were measured in lung samples from non-smokers, smokers, and patients with COPD with GOLD stage I-II and GOLD stage III-IV disease using real time RT-PCR ( n = 17–31 subjects/group). Data are mean + SD. Data were analyzed using one-way ANOVA followed by pair-wise testing with 2 tailed Student’s t-tests. *, P < 0.001 versus non-smoker group or the group indicated. In b , ADAM15 steady state mRNA levels were measured in AMs from non-smokers, smokers, and patients with COPD (all had GOLD stage I-III disease and 79% of these patients had GOLD stage I or II disease) in the BAL cohort using real time RT-PCR ( n = 6–15 subjects/group). Data are mean + SD. Data were analyzed using one-way ANOVA followed by pair-wise testing with 2 tailed Student’s t-tests. *, P < 0.001 versus non-smoker control or the group indicated. In c - d , ADAM15 and a housekeeping control (heat shock protein 90; HSP90) were quantified in AMs from non-smokers, smokers, and patients with COPD using Western blotting and densitometry. The ADAM15 levels were normalized to HSP90 levels measured in the samples and expressed as a % of the values for the non-smoker control group. The images shown in c are representative of 6–9 subjects/group. d : Data are mean + SD (n = 6–9 subjects/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with 2 tailed Student’s t-tests. *, P < 0.001 versus non-smoker control or the group indicated

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Soluble ADAM15 (sADAM15) levels in bronchoalveolar lavage fluid (BALF) and plasma samples from patients with chronic obstructive pulmonary disease (COPD), smokers, and non-smokers. a : Soluble ADAM15 (sADAM15) protein levels were measured in BALF samples from non-smokers (n = 6), smokers ( n = 19), and patients with COPD ( n = 14) using an ELISA kit. The boxes in the box-plots show the medians and 25th and 75th percentiles, and the whiskers show the 10th and 90th percentiles. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. b : sADAM15 protein levels were measured in plasma samples from non-smokers ( n = 28), smokers without COPD ( n = 27), COPD patients with GOLD stage I-II disease (n = 27), and COPD patients with GOLD stage III-IV disease ( n = 33) using an ELISA kit. The boxes in the box-plots show the medians and 25th and 75th percentiles, and the whiskers show the 10th and 90th percentiles. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: A Disintegrin and A Metalloproteinase Domain 15 (ADAM15) staining is increased in alveolar macrophages (AMs), CD8 + T cells, epithelial cells and α-SMC-positive cells in the lungs of patients with chronic obstructive pulmonary disease (COPD). Lung sections from 30 patients with COPD (4 had GOLD stage I, 10 had GOLD stage II, 4 had GOLD stage III, and 13 had GOLD stage IV disease), 10 smokers, and 10 non-smokers were double immunostained for ADAM15 and markers of macrophages (CD68; a ), CD8 + T cells (CD8; b ), airway cells staining positively for α-smooth muscle actin (α-SMA, a marker of myofibroblasts; c ), bronchial epithelial cells (pancytokeratin; d ) and alveolar epithelial cells (pancytokeratin; e ). Lung sections that were stained with non-immune isotype-matched primary antibodies showed no staining (not shown). The percentage of ADAM15-positively stained macrophages and CD8 + T cells was quantified for each cell type for each subject in 10 microscopic fields. The number of ADAM15-positively stained airway α-SMA-positive cells ( c ) and bronchial epithelial cells ( d ) and alveolar epithelial cells ( e ) was quantified and normalized to the unit area (in pixels 2 ) of airway wall (C), of bronchial epithelium ( d ) or of alveolar wall ( e ) using MetaMorph software. Data in a and b are mean + SD ( n = 10–17 subjects/group). Data were analyzed using one-way ANOVA followed by pair-wise testing with 2 tailed Student’s t-tests. *, P < 0.003 versus non-smoker control or the group indicated. In c-e , the boxes in the box-plots show the medians and 25th and 75th percentiles, and the whiskers show the 10th and 90th percentiles for 10–17 subjects/group. Data were analyzed using a Kruskal-Wallis One-Way ANOVA followed by pair-wise testing with Mann-Whitney U tests. *, P < 0.015 versus non-smoker control or the group indicated

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Staining, Marker, Software, Control, MANN-WHITNEY

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Correlations between A Disintegrin and A Metalloproteinase Domain 15 (ADAM15-positively stained) alveolar macrophages (AMs), CD8 + T cells, bronchial epithelial cells, alveolar epithelial cells, or α-SMC-positive airway cells in the lungs of patients with chronic obstructive pulmonary disease (COPD) and lung function parameters. Lung sections from 30 patients with COPD (4 were GOLD stage I, 10 were GOLD stage II, 4 were GOLD stage III, and 13 were GOLD stage IV), 9 smokers, and 10 non-smokers were double immunostained for ADAM15 and markers of macrophages (CD68), CD8 + T cells (CD8), α-smooth muscle actin; α-SMC (a marker of myofibroblasts), bronchial epithelial cells (pancytokeratin) and alveolar epithelial cells (pancytokeratin). The percentage of ADAM15-positively stained macrophages, CD8 + T cells, and the number of ADAM15-positively stained α-SMC-positive airway cells, bronchial epithelial cells or alveolar epithelial cells per unit area of airway wall, bronchial epithelial area, or alveolar wall area, respectively for each subject (on the y-axis) was quantified (as described in the legend to Fig. ) and plotted against the subject’s FEV 1 or FEV 1 /FVC values (on the x-axis). Correlations between the percentage of ADAM15-positively stained AMs and FEV 1 percent predicted or FEV 1 /FVC percent predicted are shown in a and b , respectively. Correlations between the percentage of ADAM15-positively stained CD8 + T cells and FEV 1 percent predicted and FEV 1 /FVC percent predicted are shown in c and d , respectively. Correlations between the number of ADAM15-positively stained bronchial epithelial cells per unit area of bronchial epithelium and FEV 1 percent predicted and FEV 1 /FVC percent predicted are shown in e and f , respectively. g shows the correlation between the number of ADAM15-positively stained α-SMC-positive airway cells per unit area of airway wall and FEV 1 /FVC percent predicted. All data were analyzed using the Spearman Correlation test; n = 49 subjects in each sub-figure. P < 0.05 was considered to be statistically significant

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Staining, Marker

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Correlations between A Disintegrin and A Metalloproteinase Domain 15 (ADAM15-positively stained) alveolar macrophages (AMs), CD8 + T cells, bronchial epithelial cells, alveolar epithelial cells, or α-smooth muscle actin (α-SMC)-positive airway cells and lung function or other clinical parameters. Lung sections from 31 patients with COPD (4 had GOLD stage I, 10 had GOLD stage II, 4 had GOLD stage III, and 13 had GOLD stage IV disease), 10 smokers, and 10 non-smokers were double immunostained for ADAM15 and markers of macrophages (CD68), CD8 + T cells (CD8), bronchial and alveolar epithelial cells (pancytokeratin), and α-SMC-positive small airway cells (likely myofibroblasts). The percentage of ADAM15-positively stained macrophages or CD8 + T cells in 10 microscopic fields, or the number of ADAM15-positively stained α-SMC-positive airway cells per unit area of airway wall, the number of ADAM15-positively stained bronchial epithelial cells per unit area of bronchial epithelium, or the number of ADAM15-positively stained alveolar epithelial cells per unit area of alveolar wall for each subject (on the y-axis) was quantified (as described in the legend to Fig. ) and plotted against the subject’s pack-years of smoking history, FEV 1 , or FEV 1 /FVC (on the x-axis). a-d : show the correlations between the percentage of ADAM15-positively stained AMs ( a ; n = 50 subjects), the percentage of ADAM15-positively stained CD8 + T cells ( b ; n = 50 subjects), the number of ADAM15-positively stained bronchial epithelial cells per unit area of bronchial epithelium ( c ; n = 50 subjects), the number of ADAM15-positively stained α-SMC-positive airway cells per unit area of airway wall ( d ; n = 50 subjects) and pack-years of smoking history. e shows the correlation between the number of ADAM15-positively stained α-SMC-positive airway cells per unit area of airway wall and FEV 1 percent predicted (n = 49 subjects including 10 non-smokers, 8 smokers and 31 patients with COPD). f-h : show the correlation between the number of ADAM15-positively stained alveolar epithelial cells per unit area of alveolar wall and FEV 1 percent predicted ( f ; n = 49 subjects including 10 non-smokers, 9 smokers and 30 patients with COPD), FEV 1 /FVC percent predicted ( g ; n = 49 subjects including 10 non-smokers, 9 smokers and 30 patients with COPD) and pack-years of smoking history ( h ; n = 50 subjects). All data were analyzed using the Spearman Correlation test. P < 0.05 was considered to be statistically significant

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Staining

Journal: Respiratory Research

Article Title: ADAM15 expression is increased in lung CD8 + T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction

doi: 10.1186/s12931-020-01446-5

Figure Lengend Snippet: Overexpression of A Disintegrin and A Metalloproteinase Domain 15 (ADAM15) in human THP-1 cells reduces cigarette smoke extract (CSE)-induced release of cytokines and proteinases. In a , ADAM15 steady state mRNA levels were measured in total cellular RNA samples isolated from differentiated THP-1 cells 24 h after they were electroporated with the pcDNA3.1-ADAM15 plasmid (ADAM15 over-expression [OE] vector) or a control vector (Vector), as described in Methods. In b-e , THP-1 cells were electroporated with either the ADAM15 OE or the control vector and then incubated with 5% CSE for the time indicated. Levels of MMP-9 ( b ), MMP-12 ( C ), CCL-2 ( d ) and TNF-α ( e ) protein secreted into the media were quantified using commercial ELISA kits. Data are mean + SD. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. *, P < 0.05 versus the group indicated

Article Snippet: The sections were incubated with a goat anti-human ADAM15 IgG which recognizes the ectodomain of ADAM15 (R&D Systems, Minneapolis, MN) or non-immune goat IgG, followed by Alexa 488-conjugated rabbit anti-goat fragment antigen binding-2 (F (ab) 2 ; Invitrogen, Charlestown, MA).

Techniques: Over Expression, Isolation, Plasmid Preparation, Control, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test