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: 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: 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: 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