Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 1: Tspan3 is a new ADAM10 interaction partner. (A) (I) The principle of the Split- Ubiquitin yeast two Hybrid System is depicted (modified from [27]). ADAM10 is fused to the C- terminal part of ubiquitin (Cub) and an artificial transcription factor LexA (LexA-VP16) expressed in yeast together with N-terminal NubG tagged proteins from a murine brain cDNA library. The close proximity between the ADAM10 bait protein and an interaction partner leads to the reconstituion of ubiquitin. Cellular ubiquitin proteases release the transcription factor LexA, which activates HIS3 gene transcription and allows yeast clones to grow on selective media plates lacking histidin (His). (II-III) ADAM10 bait protein was co-expressed with the identified Tspan3 (Tsp3)

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Ubiquitin Proteomics, Modification, cDNA Library Assay, Clone Assay

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 2: Tspan3 expression accelerates ADAM10 CTF generation without increasing ADAM10 surface levels. (A) Murine Tspan3-myc (Tsp3) and Tspan15-myc (Tsp15) expressed in HeLa cells (I). After immunoblotting ADAM10 was detected using an ADAM10-specific C-

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Expressing, Western Blot

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

doi: 10.1016/j.bbamcr.2016.11.003

Figure Lengend Snippet: Figure 3: Tspan3 expression leads to an ADAM10-mediated increase of the APP -CTF (C83). (A) Tspan3-myc (Tsp3) expression in HeLa cells increases the levels of the APP-CTFs but does not affect the N-Cadherin CTF production. Expressing Tspan15-myc (Tsp15) only moderately

Article Snippet: The following antibodies were used: anti-ADAM10 antibody EPR5622 (IB, Abcam, Cambridge, UK previously GenTex, Irvine, USA), anti-ADAM10 antibody raised against a the C-terminus of ADAM10 (IP, [12]), MAB946 anti-ADAM10 ectodomain (FACS, R&D Systems, Minneapolis, USA), anti-ADAM17 antibody A300D [23], anti-actin (IB, Sigma-Aldrich, Munich, Gemany), B63.2 anti-APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), anti-EEA1 (IF, Cell Signaling Technology, Danvers, USA), FL-335 anti-GAPDH (Santa Cruz Biotechnology, Dallas, USA), 9B11 anti-myc (IB and IF, Cell Signaling Technology, Danvers, USA), anti-myc (IP, GTX30518, GenetTex, Irvine, USA), anti-N-Cadherin (BD Transduction Laboratories, Heidelberg, Germany), anti-PDI (A6) (Abcam, Cambridge, UK), MAB 5232 anti-Presenilin-1 C-terminal loop (IB, Merck Millipore, Darmstadt, Germany), AB1563 anti-Presenilin-1 N-terminal (IB, Merck Millipore, Darmstadt, Germany), L2T2 anti-LIMP-2 (IF, IB [24]), anti-PDI (IF, Santa Cruz Biotechnology, Heidelberg, Germany), POM1 anti-PrP c (IB, Prof. Dr. A. Aguzzi, Zürich, Switzerland), anti-Rab5 (IF, Synaptic Systems, Göttingen, Germany), H4B4 anti-LAMP2 (IF, DSHB, Iowa Ciy, USA), anti-E7 beta-Tubulin (DSHB Iowa Ciy, USA), anti-Tspan3 (IB, IP, IF, IHC, antibody produced by Pineda, Berlin, Germany against a synthetic peptide corresponding to the last 19 amino acids of the C-terminus of mouse Tspan3).

Techniques: Expressing

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Biochemical evidence for ADAM10-mediated CA IX ECD cleavage. (A) Verification of the cleavage activity of rhADAM10 catalytic domain towards the fluorogenic peptide Mca-K-P-L-G-L-Dpa-A-R-NH 2 . The peptide was used at the final concentration of 10 µM in a total of 100 µl reaction mixture with 10 ng of the rhADAM10. The cleavage was allowed to proceed for 30, 40, 50 and 120 min. Time-related increase of the fluorescence emitted from the peptide proves that rhADAM10 was active in comparison to negative control without rhADAM10. (B) ELISA analysis of CA IX ECD in culture medium obtained from CHOwt-FL-CA IX and CHOwt-NS-CA IX cells transiently expressing FL-CA IX and NS-CA IX, respectively. Transfected cells were treated with rhADAM10 (500 ng/ml) for 24 h (Data were analyzed by Student's t-test). (C) Incubation of CHOwt-FL-CA IX cells with ADAM17 activator PMA (20 µM, 3 h), ADAM10 activator IONO (1 µg/ml, 3 h) or rhADAM10 (500 ng/ml, 24 h). Data were analyzed using one-way ANOVA followed by Dunnett's test. Experiments were performed in triplicates and repeated twice. The results were expressed as the mean ± SD. **P<0.01 and ***P<0.001. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; rhADAM10, recombinant human ADAM10; ECD, ectodomain; FL, full-length; NS, non-shed; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; ns, non-significant.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activity Assay, Concentration Assay, Fluorescence, Comparison, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Incubation, Recombinant

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Co-localization of ADAM10 and CA IX in C33a-FL-CA IX cells. Immunofluorescence of (A) C33a-FL-CA IX cells expressing CA IX, and (B) control C33a-neo cells using ADAM10-specific antibody. Nuclei were counterstained with DAPI. PLA performed in (C) C33a-FL-CA IX and (D) C33a-neo cells using rabbit anti-human CA IX-specific antibody and mouse anti-human ADAM10 antibody. Red PLA signal indicating the interaction of CA IX with ADAM10 was clearly visible only in C33a cells expressing FL CA IX. Experiment was performed in triplicates and repeated twice. Magnification, ×630. Scale bar, 10 µm. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; PLA, proximity ligation assay; FL, full-length.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Immunofluorescence, Expressing, Control, Proximity Ligation Assay

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Localization of ADAM10 (green) in response to CA9hu-1 antibody-induced CA IX internalization (red). C33a-FL-CA IX cells were pre-incubated either with anti-ADAM10 antibody alone (ctrl, A-D) or with anti-ADAM10 antibody together with the internalization-inducing anti-CA IX antibody CA9hu-1 (E-H) 30 min at 4°C. Plasma membrane staining signal for ADAM10 and CA IX was observed at all time points in the absence of CA9hu-1 pre-treatment. On the other hand, CA9hu-1-induced internalization of CA IX as well as ADAM10 was visible after 2 and 4 h at 37°C (E and F), while both molecules showed recycling to plasma membrane after 8 and 24 h. Overlapped staining signals were evident in all samples. Magnification, ×630. Scale bar, 20 µm. Experiment was performed in triplicates and repeated twice. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Incubation, Clinical Proteomics, Membrane, Staining

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: GI-induced internalization of ADAM10 and targeting of ADAM10 by RNA interference or by a dominant-negative ADAM10 mutant ∆MP. (A-H) C33a-FL-CA IX cells were pre-incubated with anti-ADAM10 antibody at 4°C. (A-D) Cells showed the plasma membrane staining signal for ADAM10 (green) in absence of GI at 37°C. (E-H) GI-induced internalization of ADAM10 was clearly visible as cytoplasmic staining signal in all incubation periods at 37°C. (I) Direct targeting of ADAM10 was performed by RNA interference and (J) expression of a dominant-negative mutant ∆MP with and without GI treatment. Control cells were transfected with either an esiRNA targeting RLUC or an empty vector (pcDNA3.1). Culture media collected from transfected cells incubated in presence and absence of GI for 24 h were collected and subjected to ELISA analysis for detection of CA IX ECD. Experiment was performed in triplicates and repeated six times. Data were analyzed by one-way ANOVA followed by Dunnett's test. Results were expressed as the mean percentage of CA IX ECD shedding with control cells set as 100% ± SD. ***P<0.001. GI, GI254023X; ADAM, a disintegrin and metalloproteinase; ∆MP, dominant-negative mutant of ADAM10; esiRNA, enzymatically-prepared small interfering RNA; CA IX, carbonic anhydrase IX; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Dominant Negative Mutation, Mutagenesis, Incubation, Clinical Proteomics, Membrane, Staining, Expressing, Control, Transfection, esiRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Additive effect of ADAM10 and ADAM17 activation/inhibition. (A) Quantitative PCR analysis of ADAM10 and ADAM17 mRNA levels in C33a-FL-CA IX and C33a-NS-CA IX cells normalized to the level of β-actin mRNA. (B) Western blot analysis of ADAM10, ADAM17 and CA IX protein in C33a-FL-CA IX and C33a-NS-CA IX cells. The anti-actin antibody was used as a loading control. (C and D) ELISA analysis of CA IX ECD in culture medium collected from C33-FL-CA IX cells after the treatment with IONO (1 µg/ml), PMA (20 µM), IONO + PMA (1 µg/ml; 20 µM), D1(A12) antibody (200 nM), GI inhibitor (1 µM) or D1(A12) + GI (200 nM; 1 µM) for 3 h in comparison to non-treated cells (CTRL). Experiment was performed in triplicates and repeated two times. Data were analyzed by (A) Student's t-test and (C and D) one-way ANOVA followed by Dunnett's test. Results were expressed as the mean relative levels of mRNA or CA IX ECD ± SD. ***P<0.001. ADAM, a disintegrin and metalloproteinase; FL, full-length; CA IX, carbonic anhydrase IX; NS, non-shed; ECT, ectodomain; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; GI, GI254023X.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Scheme illustrating CA IX ECD shedding by ADAM10 and ADAM17 proteinases. Picture depicts differences in regulatory components that differentially affect ADAMs biosynthesis and processing at various levels of their expression and activation and thereby can potentially influence CA IX ECD cleavage. Based on ( , – ). CA IX, carbonic anhydrase IX; ADAM, a disintegrin and metalloproteinase; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Expressing, Activation Assay

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 2 | Influence of metalloproteinases on the expression and release of CXCL16. (a) Analysis of soluble CXCL16 released from primary TALDCs with a CXCL16-specific ELISA. Cells were preincubated with different proteinase inhibitors for 15 min before IFN-g (24 ng/ml) was added for 24 h. Application of IFN-g (24 ng/ml) increased the amount of soluble CXCL16 in the supernatants of TALDCs. The ADAM10-specific metalloproteinase inhibitor, GI254023X (GI), and the broad-spectrum metalloproteinase inhibitors, GM6001 and TAPI-2, reduced the IFN-g-induced CXCL16 release. Data are mean±s.d. (n ¼ 3); ***Po0.001 versus control; ###Po0.001, ##Po0.01; #Po0.05 versus cytokine-treated cells. (b) Inhibition of metalloproteinases increased cellular CXCL16 protein expression in TALDCs. Fifteen minutes before application of IFN-g, cells were pretreated with the indicated concentration of the metalloproteinase inhibitors GI254023X, GM6001, and TAPI-2. CXCL16 protein levels were measured by western blot analysis and b-actin was used as a loading control. (c) Knockdown of CXCL16 with CXCL16-specific siRNA in TALDCs is shown by western blot analysis. (d) Application of pharmacological inhibitors of metalloproteinases increased the amount of cellular CXCL16. Cell lysates were isolated as described under Materials and Methods, and 15 mg total proteins were used to measure the amount of cell-expressed CXCL16 by CXCL16-specific ELISA. Data are mean þ s.d. (n ¼ 5). Statistically significant release of CXCL16 (Po0.001) is indicated by asterisk. (e) Suppression of ADAM10 protein levels by RNA interference in the presence and absence of IFN-g. Cell lysates of TALDCs were prepared 48 h after transfection with siRNA specific for ADAM10 and an unspecific siRNA. Mock-transfected cells treated with the transfection lipid in the absence of siRNA were used as a control. Western blot analysis of ADAM10 protein expression was performed. Blots were reprobed with an antibody specific for b-actin as a loading control. (f) Inhibition of ADAM10 by siRNA reduced the IFN-g-stimulated CXCL16 release in TALDCs. Supernatants of siRNA-treated and IFN-g- stimulated or unstimulated cells were analyzed for soluble CXCL16 using a CXCL16-specific ELISA (n ¼ 3); ***Po0.001 versus control; ###Po0.001 versus IFNg-treated cells.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Inhibition, Concentration Assay, Western Blot, Knockdown, Isolation, Transfection

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 3 | Localization of ADAM10 protein in normal human kidney. (a) In the overview, ADAM10 protein is mainly expressed in tubular cells, but expression was also found in glomerular cells. Serial sections of normal renal tissue represented constitutive ADAM10 expression in aquaporin-2 (e), calbindin D-28K (f), and Tamm–Horsfall glycoprotein (g) expressing tubular profiles, which correspond to CD, (DCT, CNT), and TAL, respectively. Proximal tubules were devoid of any specific ADAM10 staining. (h) Immunofluorescent detection of ADAM10 (red fluorescence) in aquaporin-2-positive principal cells of the CD (green fluorescence). A prominent overlap of both signals in the same cells can be seen, indicated in the examples by asterisks. In contrast, arrows indicate cells negative for either aquaporin-2 or ADAM10, thus representing presumable intercalated cells. (i) ADAM10 is not expressed in the intercalated cells of the CD. Note: arrows represent H þ-ATPase-expressing intercalated cells (green colour) that do not express ADAM10 (red colour); compare with the merged view. (j) Confocal immunofluorescence analysis of ADAM10 and CXCL16 expression in a cortical CD. Tissue section was stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. Both signals can be confined to same segment-specific cells; see merged picture on the right.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Staining, Fluorescence, Immunofluorescence

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 4 | Supernatants from ADAM10- and CXCL16 siRNA-transfected TALDCs decreased the chemotaxis of Jurkat T cells. (a) Surface expression of the chemokine receptor CXCR6 in Jurkat T cells analyzed by fluorescence-activated cell sorting analysis. (b) Inhibition of CXCL16 protein expression by siRNA fully abolished release of CXCL16 in TALDCs. Cells were transfected with different siRNA duplexes for inhibition of ADAM10 or CXCL16, an unspecific siRNA (scrambled), or not transfected with siRNA (mock). A total of 24 h after transfection, TALDCs were stimulated with IFN-g for 24 h, where indicated, and supernatants were collected. Soluble CXCL16 was determined by a CXCL16-specific ELISA (n ¼ 4). Data are mean±s.d.; ***Po0.001 versus control; ###Po0.001 versus IFN-g-treated cells. (c) Decrease of soluble CXCL16 correlates with a reduction in the migration of Jurkat T cells. Supernatants shown in Figure 5b were used for chemotaxis assays (n ¼ 2). Recombinant CXCL16 induced migration of CXCR6-expressing Jurkat T cells about threefold; **Po0.01; *Po0.05 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Chemotaxis Assay, Expressing, Fluorescence, FACS, Inhibition, Enzyme-linked Immunosorbent Assay, Control, Migration, Recombinant

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 5 | Increased urinary CXCL16 correlated with focally apical CXCL16 expression in tubular cells of renal allografts with the histopathological diagnosis of ATN. Six allograft biopsies of patients diagnosed with ATN (cases A1–A6; clinical parameters and CXCL16 analysis are shown in Table 1) were analyzed by CXCL16 immunohistochemical analysis (a–f) and with ADAM10 (green colour) and CXCL16 (red colour) double immunofluorescence staining (g–l). Notably, all patients showed focally increased apical CXCL16 expression in renal tubuli. Strongest apical CXCL16, seen in patient A4 (d), correlated with the highest amount of urinary CXCL16 (Table 1). In contrast, ADAM10 expression (green colour) was not significantly changed in ATN patients (A1–A6) compared with normal kidney (g–l). (m) Urinary CXCL16 measured by a CXCL16-specific ELISA in healthy volunteers (normal), in patients with IR, and in patients with ATN. Data are mean±s.d.; **Po0.01 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Double Immunofluorescence Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 6 | Increased ADAM10 expression in allograft biopsies of kidney transplant patient with the clinical and histopathological diagnosis of acute IR. Renal allograft biopsies of patients diagnosed with IR were analyzed by double immunofluorescence for ADAM10 and CXCL16 expression in comparison with normal kidney (upper panel). Clinical features and ADAM10 analysis of patients are listed in Table 2. Tissue sections were stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. In some tubuli of patient B1 (marked with arrows), strong basolateral ADAM10 expression and no CXCL16 expression could be seen (middle panel). In contrast, in few tubuli of the same patient (marked with a star) both molecules colocalized. In the lower panel (patient B5), strong coexpression of ADAM10 and CXCL16 in a tubule is visible, accompanied with interstitial inflammatory infiltrates.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunofluorescence, Comparison, Staining

Journal: Molecular Medicine

Article Title: Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

doi: 10.1186/s10020-024-00953-1

Figure Lengend Snippet: Western blot antibodies

Article Snippet: ADAM10 , Rat anti-mouse , 1:500 , R&D System (MAB-946-100).

Techniques: Western Blot

Journal: Molecular Medicine

Article Title: Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

doi: 10.1186/s10020-024-00953-1

Figure Lengend Snippet: Immunocytochemistry antibodies

Article Snippet: ADAM10 , Rat anti-mouse , 1:500 , R&D System (MAB-946-100).

Techniques: Immunocytochemistry

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