goat anti adam15 af935 antibody  (R&D Systems)

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Differential expression of lncRNAs between ADAM15-expressing and non-expressing synovial fibroblasts (SF) under mechanical strain. ( A – D ) SF ( n = 4), either expressing ADAM15 or downregulated with a specific siRNA, were strained for 3 h using the Flexcell System (elongation 15%, frequency 1 Hertz). Reverse-transcribed cDNAs were then amplified in Arraystar lncRNA PCR-plates, and the fold change of up-/down-regulation of gene expression was calculated with the 2 −∆∆Ct method. ( A,C ) Top 20 lncRNAs differentially up- and downregulated in ADAM15-expressing versus non-expressing SF. ( B,D ) Venn diagram of all differentially expressed lncRNAs with a 2-fold change up/downregulation, identifying HOTAIR and H19-2 as differentially downregulated lncRNAs in all 4 SF (underlined in C ; intersection, boxed in D ). Intersections among 3 donors are shown in grey.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Quantitative Proteomics, Expressing, Reverse Transcription, Amplification, Gene Expression

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: ADAM15-dependent downregulation of HOTAIR under mechanical strain, resulting in the upregulation of SIRT1. ( A – D ) SF with prior downregulation of ADAM15 were strained for 0–9 h, and HOTAIR and SIRT mRNA and protein expression was quantified by qPCR and immunoblotting. ( A ) GAPDH-normalized Ct values for HOTAIR from one representative donor, showing higher Ct values, i.e., reduced HOTAIR levels, in ADAM15-expressing SF (dashed line), as compared to SF treated with ADAM15 siRNA (solid line). ( B,C ) Fold change (mean ± SD from 7 donors) of HOTAIR and SIRT1 mRNA in ADAM15-expressing versus non-expressing SF. *** p < 0.0005, by Student’s t -test, when comparing stimulated versus unstimulated HOTAIR/SIRT levels. ( D ) Immunoblots from SF silenced with ADAM15 siRNA (I) and negative control siRNA (N), showing increased SIRT1 expression in ADAM15-expressing cells after 6 h strain. ( E,F ) Fold change of SIRT1 after HOTAIR downregulation in SF ( n = 6) when unexposed to mechanical strain by siRNA and negative control siRNA (N), showing increased SIRT1 mRNA ( E ) and protein levels ( F ). Tubulin served as a loading control.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Expressing, Western Blot, Negative Control, Control

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Impact of ADAM15 and SIRT1 on histone acetylation, ROS and NAD+ in mechanically strained SF. ( A–C ) ADAM15 was downregulated by siRNA and a non-silencing siRNA (neg) as control. ( A ) Immunoblots from nuclear and cytoplasmic lysates of SF, showing decreased deacetylated histone after 6 and 9 h strain in ADAM15-expressing cells only. Tubulin and histone deacetylase (HDAC1) served as loading controls. ( B,C ) ROS and NAD+ assays in ADAM15-expressing SF (neg control) as compared to ADAM15-silenced cells. Each symbol represents the mean value of one individual donor, the horizontal bar (-) the median of 6 different donors. * p < 0.05, by Wilcoxon signed-rank test for comparison of ADAM15-expressing versus non-expressing SF. ( D ) ROS and NAD+ assays from mechanically strained SF with prior downregulation of SIRT1 by siRNA (I) and a non-silencing siRNA (N) from one representative donor. *** p < 0.0005, by Student’s t -test for SIRT1-expressing versus non-expressing SF. ( E ) ROS and NAD+ assays from strained SF in the presence of SIRT1 inhibitor selisistat (0 µM; solid line, 50 µM; double line and 100 µM; dashed line) from one representative donor. *** p < 0.0005, by Student’s t -test, comparing DMEM with the inhibitor. Representative results of at least three independent experiments are shown.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Control, Western Blot, Expressing, Histone Deacetylase Assay, Comparison

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Impact of JNK inhibition on ADAM15-mediated HOTAIR and SIRT1 regulation by mechanosignaling. ( A ) Immunoblots of SF mechanically strained for 30 and 60 min, with prior downregulation of ADAM15 by siRNA (I) and non-silencing siRNA (N) as control, showing ADAM15-dependent activation of Src, FAK and JNK. ( B ) Immunoblots of SF strained in the presence of the JNK inhibitor SP600125 or the Src inhibitor dasatinib. Tubulin served as a loading control. ( C ) Fold change of HOTAIR and ( D ) SIRT1 mRNA levels, calculated by the 2 −∆∆Ct method, comparing DMEM control with the respective inhibitor. Mean values ± SD from 6 different donors are shown.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Inhibition, Western Blot, Control, Activation Assay

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Strain-induced ATP release is dependent on ADAM15 and calcium signaling. ( A ) ATP release and ( B ) total ATP of SF strained for 9 h with prior downregulation of ADAM15 by siRNA and negative siRNA as control. Each dot represents the mean value of one individual donor, the horizontal bar (-) the median of 7 different donors. * p < 0.05 by Wilcoxon signed-rank test, comparing ADAM15-expressing versus non-expressing SF. ( C ) ATP release and ( D ) total ATP from SF stimulated with DMEM and inhibitors of TRPV4, CaM, JNK or SIRT1. *** p < 0.0005, by Student’s t -test, when comparing DMEM with the inhibitor. Representative results out of at least three independent experiments are shown.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Control, Expressing

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: ATP upregulates the expression of ADAM15. Western blot analysis of ADAM15 expression in RASFs stimulated with ATP-γ-S (200 µM) for 48 h. Tubulin served as a loading control. Representative results out of three independent experiments are shown. Densitometric evaluation of protein bands, normalized to tubulin, are shown as numbers above the gel lanes.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Strain-induced ATP release is dependent on activated pannexin-1 (PANX1). ( A ) Immunoblots from SF, strained for 0–9 h, with prior downregulation of ADAM15 by siRNA (I) or non-silencing siRNA (N), showing increased phosphorylations of PANX1 and Src in ADAM15-expressing SF. ( B ) Immunoblots of strained SF in the presence of dasatinib. ( C ) ATP release and ( D ) total ATP of strained SF in the presence of dasatinib (1 µM) and the PANX1 channel inhibitor carbenoxolone (carbenoxo, 100 µM). Data show the mean ±SD from one representative experiment out of at least 3 independent experiments. *** p < 0.0005, by Student’s t -test, for comparison of DMEM with inhibitor-treated SF. ( E ) ATP release and ( F ) total ATP from SF with downregulated ADAM15 (siA15), double knockdown of ADAM15/HOTAIR (siA15+Hot), single knockdown of HOTAIR (siHot), and negative siRNA (Neg). ** p < 0.005; *** p < 0.0005, using Student’s t -test.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Western Blot, Expressing, Comparison, Knockdown

Journal: Cells

Article Title: A Novel Pro-Inflammatory Mechanosensing Pathway Orchestrated by the Disintegrin Metalloproteinase ADAM15 in Synovial Fibroblasts

doi: 10.3390/cells10102705

Figure Lengend Snippet: Interaction of ADAM15 with transient receptor potential vanilloid 4 (TRPV4) channel and colocalization in the cell membrane. ( A,B ) Immunoprecipitations (IP) of ( A ) SF with downregulated ADAM15 by siRNA (I) or non-silencing siRNA (N) and ( B ) chondrocyte cell line transfected with full-length ADAM15 (+) or ADAM15 lacking the cytoplasmic domain (∆C), using TRPV4 or ADAM15 antibodies, or IgG as control. GAPDH served as a loading control. wcl—whole-cell lysate. ( C ) Confocal microscopy of double immunofluorescence stainings of SF, using ADAM15 and TRPV4 antibodies. Objective 40x, size bar = 20 μm. The white box marks the magnified area. ( D ) Immunoblots of the cell surface biotinylated and purified membrane fractions of SF with ADAM15-silenced by siRNA (I) or non-silencing siRNA (N), showing TRPV4 in ADAM15-expressing cell membranes only. Co = non-biotinylated cell lysates, purified on streptavidin magnetic beads, served as the background control (N). Representative results out of three independent experiments are shown. ( E ) Diagram of summarized results: cyclic strain results in ADAM15-mediated activation of JNK, the downregulation of HOTAIR and subsequent upregulation of SIRT1, leading to decreased ROS, increased NAD+ levels and ATP release. In parallel, ADAM15-mediated Src activation results in the phosphorylation of PANX1, thereby activating PANX1-mediated ATP release. The interaction with ADAM15 inhibits the constitutive cycling of the mechanosensitive calcium channel TRPV4 to the endosome (dashed double arrow). The knockdown of ADAM15 not only blocks mechanical force-induced JNK- and HOTAIR-dependent upregulation of SIRT1 completely but also eliminates all respective downstream effects on NAD+, ROS and ATP, including its release as a purinergic mediator of inflammation. ECM—extracellular matrix.

Article Snippet: Goat-anti-ADAM15 AF935 antibody was from R&D Systems; Wiesbaden, Germany.

Techniques: Membrane, Transfection, Control, Confocal Microscopy, Immunofluorescence, Western Blot, Purification, Expressing, Magnetic Beads, Activation Assay, Phospho-proteomics, Knockdown

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: Fig. 1 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: Fig. 2 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: Fig. 3 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 pixels2) 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: Fig. 4 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. 3) and plotted against the subject’s FEV1 or FEV1/FVC values (on the x-axis). Correlations between the percentage of ADAM15-positively stained AMs and FEV1 percent predicted or FEV1/FVC percent predicted are shown in a and b, respectively. Correlations between the percentage of ADAM15-positively stained CD8+ T cells and FEV1 percent predicted and FEV1/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 FEV1 percent predicted and FEV1/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 FEV1/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: Fig. 6 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

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Anti-apoptotic property of full-length ADAM15. A, ADAM15-, ADAM15Δcyto-, and vector-transfected cells were treated with 20 μm camptothecin and caspase-3/7 activity as a marker for apoptosis was measured for 0–10 h. B, the ATP content, which correlates with cell viability, was determined after exposure of the cells for 18 h using increasing camptothecin concentrations. Both assays show that the removal of the cytoplasmic tail of ADAM15 resulted in a loss of the anti-apoptotic and cell protective capacity compared with chondrocytes transfected with full-length ADAM15. *, p < 0.0004, when comparing the caspase activity or viability of the ADAM15- versus vector-transfected cells.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Plasmid Preparation, Transfection, Activity Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Enhanced phosphorylation of FAK in ADAM15-transfected chondrocytes after apoptosis induction by camptothecin. A, immunoblots of cell lysates of vector-transfected (−) and ADAM15-transfected cells (+) treated with 2 μm camptothecin for up to 60 min, showing a stronger phosphorylation at Tyr-576, Tyr-861, and Tyr-397 of FAK in the ADAM15-expressing cells in comparison with vector-transfected cells. B, T/C28a4 cells transfected with ADAM15 lacking the cytoplasmic tail (ΔC), however, did not display an activation of FAK. Blots were controlled for ADAM15 and/or ADAM15ΔC expression, and loading was monitored using anti tubulin antibodies. The immunoblots are representative of at least five repeated experiments.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Transfection, Western Blot, Plasmid Preparation, Expressing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Generation of recombinant ADAM15 and FAK proteins for protein binding assays. A, upper left panel: Coomassie stained SDS-PAGE of the purified Gst-tagged FAK fragments and the Gst- and Myc-tagged cytoplasmic domain of ADAM15 (cyto) and upper right panel: the detection of these proteins with anti-Gst antibodies by Western blotting. A, lower panels: SDS-PAGE of the cytoplasmic domain of ADAM15 with the Gst tag cleaved off by PreScission Protease, followed by immunodetection using anti-Myc and anti-ADAM15 antibodies. B, pulldown assays: cell lysates spiked with Myc-tagged ADAM15 were co-incubated with either of the three different FAK proteins. Upon immunoprecipitation (IP) using anti-Gst-Sepharose and subsequent immunoblotting (IB), bound ADAM15 was detected by anti-Myc antibodies. Blots were stripped, and the Gst-tagged FAK proteins were verified by anti-Gst antibodies, thereby demonstrating the interaction of ADAM15 with the C-terminal FAK-region (amino acids 707–913).

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Recombinant, Protein Binding, Staining, SDS Page, Purification, Western Blot, Immunodetection, Incubation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Interaction of ADAM15 with FAK. A, the domain structure of FAK: the FERM, the catalytic domain, and the C-terminal domain with the FAT domain. Black bars, two proline-rich regions (PXXP motifs) are binding sites for SH2/SH3 domain containing proteins. B and C, mammalian two-hybrid assay. The distinct FAK domains, cloned as segregated fragments into the pCMV-BD bait vector, were co-transfected with the cytoplasmic domain of ADAM15 in pCMV-AD prey vector and a firefly luciferase reporter into HEK293 cells. B, an exclusive interaction of ADAM15 with a C-terminal region of FAK (amino acids 707–913), but not with the FERM, the kinase, or the FAT domain is demonstrated. Mutation of Tyr-861 into Phe-861 in the interaction domain of FAK had no influence on ADAM15 binding. C, left panel, further fine-mapping of the Fak domain 707–913 revealed fragment 730–790 (underlined) as the smallest region with binding capacity equivalent to 707–913. Shortening of 10 amino acids either at the N or C terminus abrogates the binding to ADAM15 by ∼70%. Shown is a representative experiment. C, right panel, depicts the mean (± S.D.) of five repeated assays of ADAM15 binding with each distinct FAK fragment. Measured luciferase values were normalized to the values obtained from a co-transfected Renilla luciferase control plasmid.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Binding Assay, Two Hybrid Assay, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Co-localization of ADAM15 and FAK. ADAM15 transfected chondrocytic T/C28a4 cells (A) and human osteoarthritic chondrocytes (B) were grown on collagen type II and double-stained with goat anti-ADAM15 and mouse anti-FAK antibodies visualized with Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies using confocal laser scanning microscopy (40× objective). C, shown is a magnification of the white boxed area in B with co-localized spots marked by white arrows. The merged images clearly reveal a co-localization of ADAM15 and FAK in focal contacts.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Transfection, Staining, Confocal Laser Scanning Microscopy

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Stronger phosphorylation of Src kinase in ADAM15-transfected cells is dependent on direct binding to FAK. A, upon stimulation with camptothecin for 60 min, an enhanced phosphorylation of Tyr-416 Src was detected in whole cell lysates of ADAM15-transfected cells (+) as compared with those derived from respective vector control cells (−) by immunoblotting. B, to analyze whether the stronger phosphorylation of Src results from an interaction with ADAM15 or with FAK, co-immunoprecipitations (IP) were performed by using as the precipitating antibody either 1) anti-ADAM15 or 2) anti FAK. C, lysates of ADAM15 (+) and vector control cells (−) upon camptothecin exposure were immunoprecipitated with either anti-ADAM15 1) or anti-FAK antibodies 2) and immunoblotted using anti Tyr-416 Src antibodies. A stronger phosphorylation of Tyr-416 Src was observed in the ADAM15-transfected cells compared with the vector control cells, independent of the antibody used for the precipitation. Blots were stripped, and precipitated Src was visualized using anti-Src antibodies. Src was pulled down in ADAM15 precipitated lysates only (vector-transfected cells, negative). In all anti-FAK antibody-precipitated lysates, equal amounts of Src protein were demonstrated. Blots were restripped and controlled for precipitated ADAM15 1) or FAK 2) using respective antibodies. D, mammalian two-hybrid was used to analyze the binding partner of Src. The N-terminal SH2/SH3 domain as well as full-length Src were cloned into the prey vector and distinct FAK domains into bait vector. Bait and prey were co-transfected with the firefly luciferase reporter and a Renilla luciferase control plasmid into HEK cells. A strong binding of the SH2/SH3 domain as well as full-length Src to the C-terminal region of FAK (707–913) was measured, whereas the FERM, the catalytic, and the FAT domain did not bind to Src. Mutation of Tyr-861 into Phe-861 of FAK did not affect Src interaction. However, no direct binding of ADAM15 to SH2/SH3 Src or full-length Src could be detected by contrast to the already proven interaction with the C-terminal FAK domain (707–1052, Fig. 4) that served as a positive control in this experiment.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Transfection, Binding Assay, Derivative Assay, Plasmid Preparation, Western Blot, Immunoprecipitation, Clone Assay, Luciferase, Mutagenesis, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Signaling of ADAM15 expressed as a IL-2 receptor-α/cytoADAM15 chimera. The T/C28a4 chondrocyte cell line was stably transfected with a chimeric construct composed of the extracellular IL-2 receptor-α (CD25) fused to the cytoplasmic tail of ADAM15, and the chimeric protein was detected by immunoblotting using either anti-CD25 or anti-ADAM15 antibodies at ∼60 kDa (A, left panels). A, right panel, shows co-immunoprecipitation of FAK only in cell lysates of cells transfected with the chimeric IL2Rα/cytoADAM15 construct (Ch) as compared with vector transfected cells (−) using anti-CD25 antibodies. B, immunoprecipitation (IP) using anti-FAK antibodies show co-precipitated Src in both vector and chimera-transfected cells, but an enhanced phosphorylation of Tyr-416 Src is noted exclusively in the IL2Rα/cytoADAM15 cells following stimulation with IL-2. C, immunoblots (IB) of lysates derived from IL-2 stimulated cells exhibit a stronger phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 in IL2Rα/cytoADAM15 cells. Equal loading was controlled by reprobing with anti-FAK antibodies.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Stable Transfection, Transfection, Construct, Western Blot, Immunoprecipitation, Plasmid Preparation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Inhibition of FAK and Src signaling by FAK 14 inhibitior and PP2 in the presence of ADAM15. Vector- (black bars) and ADAM15-transfected cells (open bars) were treated with 0.25 μm and 0.5 μm FAK 14 inhibitor (A) and 5 nm and 10 nm PP2 (B) for the time points shown, and caspase-3/7 activity was determined. A significantly increased caspase activity was detected in the control cells in contrast to ADAM15-transfected cells that exhibited higher apoptosis resistance to both inhibitors. Incubation with DMEM alone also displayed a significantly higher caspase activity in vector control cells. C and D, ADAM15 transfected cells were silenced using siRNA I and II for 40 h and treatment with 0.25 μm FAK 14 inhibitor (C) or 5 nm PP2 (D) resulted in a significantly higher caspase activity as compared with cells silenced with a nonfunctional siRNA (N) or with transfection agent alone (0). Incubation with DMEM alone served as a control. Shown are representative results of least five repeated experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Inhibition, Plasmid Preparation, Transfection, Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: ADAM15 Protein Amplifies Focal Adhesion Kinase Phosphorylation under Genotoxic Stress Conditions *

doi: 10.1074/jbc.M112.347120

Figure Lengend Snippet: Enhanced phosphorylation of FAK and Src in osteoarthritic chondrocytes after apoptosis induction by camptothecin. Immunoblots of cell lysates of OA chondrocytes that were silenced using ADAM15-specific siRNAs I and II and a nonfunctional siRNA (N) or transfection agent alone (0) and treated with camptothecin, showing marked induction of phosphorylation of FAK at Tyr-576, Tyr-861, and Tyr-397 and Src at Tyr-416 by camptothecin after 15 and 30 min (N and 0), which is strongly reduced after silencing of ADAM15 (I and II). Shown are representative results of an analysis performed on primary chondrocytes derived from five OA cartilage samples.

Article Snippet: 1 × 10 4 OA chondrocytes and ADAM15-transfected cells were seeded into the chamber slides and grown for 24 h. After fixation in 4% paraformaldehyde in PBS and blocking with PBS/1% BSA, cells were stained with goat anti-ADAM15 (1:100) and mouse anti-FAK (1:100) for 2 h and visualized with Alexa Fluor 488 anti-goat and Alexa Fluor 594 anti-mouse conjugated antibodies (1:200; Molecular Probes) using confocal laser scanning microscopy.

Techniques: Western Blot, Transfection, Derivative Assay