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Millipore rabbit polyclonal antibody against α-disintegrin and metalloproteinase 10 (adam10)
Rabbit Polyclonal Antibody Against α Disintegrin And Metalloproteinase 10 (Adam10), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+polyclonal+antibody+against+adam10/anti+adam10/pm30012417-102-17-21
Average 90 stars, based on 1 article reviews

rabbit polyclonal antibody against α-disintegrin and metalloproteinase 10 (adam10) - by Bioz Stars, 2026-09

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Article Title: Anxiety-like but not despair-like behaviors are further aggravated by chronic mild stress in the early stages of APP swe /PS1dE9 transgenic mice
Article Snippet: .. After being blocked with 5% milk, the bands were incubated with rabbit polyclonal antibody against ADAM10 (1:1000; Millipore), ASC (1:1500; Santa Cruz), BDNF (1:300; Abcam), CREB (1:1000; Cell Signaling Technology), Caspase1 (1:1000; Millipore), IDE (1:800; Abcam), IL-1β (1:1000; Millipore), IL-6 (1:1000; Abcam), NEP (1:800; Millipore), PS1 (1:1000; Sigma), NLRP3 (1:1000; AdipoGen), p-CREB (1:1000; Cell Signaling Technology), procaspase 1 (1:500; Millipore), sAPPα (1:800; IBL), TrkB (1:500; Santa Cruz), TNF-α (1:1000; Abcam), rabbit monoclonal antibody against Aβ 1-42 (1:1000; Abcam), or mouse monoclonal antibody against BACE1 (1:1000; Millipore) at 4°C overnight. .. Following TBST washing, bands were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000; Vector Laboratories), and then visualized with ECL plus detection system.

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Pathogen-specific regulation of a disintegrin and metalloproteinase (ADAM)10 protein expression and surface localization in bacterial infection. A549 cells were grown to confluence and either left unstimulated or infected with Pseudomonas aeruginosa (P. aeruginosa ) (multiplicity of infection of 5 (MOI 5) ( A , B ), infected with Streptococcus pneumoniae ( S. pneumoniae ) (MOI 5) ( C , D ) or stimulated with exotoxin A (ExoA) (100 ng/mL, E , F ). In ( A – E ), samples were taken after an incubation time of 30, 60, 120 or 240 min. In ( F ), samples were probed after 4 h. ( A , C , E ): ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against glyceraldehyde-3-phosphat dehydrogenase (GAPDH) served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A , right) (antibody specificity detailed in ). ( B , D , E ): ADAM10 surface expression was investigated by surface staining with an N-terminal antibody against ADAM10 (1 µg/mL) and an APC-coupled secondary antibody (5 µg/mL) and subsequent flow cytometric analysis (quantification as mean fluorescence intensity). The values of the adequate isotype control were subtracted followed by normalization to the unstimulated cells. A representative histogram is shown in ( B , left). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference to the control calculated using two tailed two samples t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific regulation of a disintegrin and metalloproteinase (ADAM)10 protein expression and surface localization in bacterial infection. A549 cells were grown to confluence and either left unstimulated or infected with Pseudomonas aeruginosa (P. aeruginosa ) (multiplicity of infection of 5 (MOI 5) ( A , B ), infected with Streptococcus pneumoniae ( S. pneumoniae ) (MOI 5) ( C , D ) or stimulated with exotoxin A (ExoA) (100 ng/mL, E , F ). In ( A – E ), samples were taken after an incubation time of 30, 60, 120 or 240 min. In ( F ), samples were probed after 4 h. ( A , C , E ): ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against glyceraldehyde-3-phosphat dehydrogenase (GAPDH) served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A , right) (antibody specificity detailed in ). ( B , D , E ): ADAM10 surface expression was investigated by surface staining with an N-terminal antibody against ADAM10 (1 µg/mL) and an APC-coupled secondary antibody (5 µg/mL) and subsequent flow cytometric analysis (quantification as mean fluorescence intensity). The values of the adequate isotype control were subtracted followed by normalization to the unstimulated cells. A representative histogram is shown in ( B , left). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference to the control calculated using two tailed two samples t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Mouse monoclonal antibody against human CD9 (MM2/57); Rabbit polyclonal antibody against human ADAM10 C-terminus was obtained from Invitrogen (Frankfurt, Germany), rabbit polyclonal antibody against human GAPDH from Santa Cruz Biotech (Dallas, TX, USA).

Techniques: Expressing, Infection, Incubation, Western Blot, Staining, Fluorescence, Two Tailed Test

P. aeruginosa and ExoA promote ADAM10 activation and shedding activity. ( A – C ) A549 cells were transfected with a plasmid encoding for alkaline phosphatase (AP)-coupled betacellulin (AP-BTC) and seeded at equal density. Cells were pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left unstimulated or infected with P. aeruginosa in ( A ) (MOI 5 for 2 and 4 h), stimulated with ExoA in ( B ) (100 ng/mL for 4 h), or infected with S. pneumoniae in ( C ) (MOI 5 for 4 h). Finally, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. ( D , E ) A549 cells were either pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left unstimulated and either infected with P. aeruginosa in ( D ) (MOI 5 for 2 and 4 h) or stimulated with ExoA in ( E ) (100 ng/mL, for 4 h). After the mentioned stimulation time, cells were lysed and cleavage of E-cadherin was investigated by Western blot, probing with antibodies against the C-terminus of E-cadherin followed by beta actin as loading control. Representative blots of three independent experiments are shown. Quantitative data ( A – C ) are shown as means + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using two-way ANOVA and Tukey post-test (** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant).

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: P. aeruginosa and ExoA promote ADAM10 activation and shedding activity. ( A – C ) A549 cells were transfected with a plasmid encoding for alkaline phosphatase (AP)-coupled betacellulin (AP-BTC) and seeded at equal density. Cells were pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left unstimulated or infected with P. aeruginosa in ( A ) (MOI 5 for 2 and 4 h), stimulated with ExoA in ( B ) (100 ng/mL for 4 h), or infected with S. pneumoniae in ( C ) (MOI 5 for 4 h). Finally, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. ( D , E ) A549 cells were either pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left unstimulated and either infected with P. aeruginosa in ( D ) (MOI 5 for 2 and 4 h) or stimulated with ExoA in ( E ) (100 ng/mL, for 4 h). After the mentioned stimulation time, cells were lysed and cleavage of E-cadherin was investigated by Western blot, probing with antibodies against the C-terminus of E-cadherin followed by beta actin as loading control. Representative blots of three independent experiments are shown. Quantitative data ( A – C ) are shown as means + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using two-way ANOVA and Tukey post-test (** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant).

Techniques: Activation Assay, Activity Assay, Transfection, Plasmid Preparation, Incubation, Infection, Western Blot

Function of ADAM10 in P. aeruginosa induced protein permeability and leukocyte transmigration. A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr) as indicated in the graph legend. Cells were grown in trans wells until confluence. Cells were pre-incubated with 0.1% DMSO (vehicle control) or with ADAM10 inhibitor GI254023X (10 μM, black bars). ( A , B ) Cells were either left unstimulated or infected with P. aeruginosa in ( A ) (MOI 5 for 4 h) or stimulated with ExoA in ( B ) (100 ng/mL for 4 h). Subsequently, the cell culture medium in the upper chamber was replaced by 70-kDa TRITC dextran and FITC-albumin suspension in PBS supplemented with 0.2 % BSA, and the permeability was measured by TRITC-dextran and FITC-albumin diffusion into the lower wells. Paracellular permeability is shown as percentage, calculated in relation to the background empty transwell (100%). ( C , D ) Cells were either left unstimulated, infected with P. aeruginosa in ( C ) (MOI 5 for 4 h) or stimulated with ExoA in ( D ) (100 ng/mL for 4 h). Subsequently, 2 × 10 5 THP-1 cells were added to the upper chamber in the presence or absence of human CCL-2 (3 nM) as chemoattractant for monocytes. After 45 min, the number of transmigrated cells was determined by measurement of endogenous β-glucoronidase activity in the lower chamber. Quantitative data are shown as mean + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using one-way ANOVA and Tukey post-test in ( A , B ). In ( C , D ), asterisks and rhombs indicate significance among treated cells in the absence or presence of CCL2, respectively, calculated using two-way ANOVA and Tukey post-test (*/# p < 0.05, **/## p < 0.01, *** p < 0.001, **** p < 0.0001).

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Function of ADAM10 in P. aeruginosa induced protein permeability and leukocyte transmigration. A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr) as indicated in the graph legend. Cells were grown in trans wells until confluence. Cells were pre-incubated with 0.1% DMSO (vehicle control) or with ADAM10 inhibitor GI254023X (10 μM, black bars). ( A , B ) Cells were either left unstimulated or infected with P. aeruginosa in ( A ) (MOI 5 for 4 h) or stimulated with ExoA in ( B ) (100 ng/mL for 4 h). Subsequently, the cell culture medium in the upper chamber was replaced by 70-kDa TRITC dextran and FITC-albumin suspension in PBS supplemented with 0.2 % BSA, and the permeability was measured by TRITC-dextran and FITC-albumin diffusion into the lower wells. Paracellular permeability is shown as percentage, calculated in relation to the background empty transwell (100%). ( C , D ) Cells were either left unstimulated, infected with P. aeruginosa in ( C ) (MOI 5 for 4 h) or stimulated with ExoA in ( D ) (100 ng/mL for 4 h). Subsequently, 2 × 10 5 THP-1 cells were added to the upper chamber in the presence or absence of human CCL-2 (3 nM) as chemoattractant for monocytes. After 45 min, the number of transmigrated cells was determined by measurement of endogenous β-glucoronidase activity in the lower chamber. Quantitative data are shown as mean + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using one-way ANOVA and Tukey post-test in ( A , B ). In ( C , D ), asterisks and rhombs indicate significance among treated cells in the absence or presence of CCL2, respectively, calculated using two-way ANOVA and Tukey post-test (*/# p < 0.05, **/## p < 0.01, *** p < 0.001, **** p < 0.0001).

Techniques: Permeability, Transmigration Assay, Transduction, shRNA, Incubation, Infection, Cell Culture, Diffusion-based Assay, Activity Assay

Differential function of ADAM10 in epithelial regeneration. A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr), as indicated in the graph legend. Cells were grown in 96-well plates until confluence and treated for 2 h with mitomycin (5 µg/mL) to avoid cell proliferation. In ( A , B ), cells were pre-incubated with 0.1% DMSO (vehicle control) or with ADAM10 inhibitor GI254023X (10 μM). Subsequently, cells were either left unstimulated or stimulated with ExoA (100 ng/mL). After 4 h, the stimulant was removed and the cells were investigated for wound closure after automated scratch induction over a period of 24 h using a live cell imaging system. In ( C , D ), the same setup was used. Additionally, GI254023X was added after ExoA treatment and scratch induction. Data are shown as a percentage of wound closure relative to control treated cells ( A , C representative images; B , D automated quantifications). Quantitative data are shown as means + SD of six independent experiments. Asterisks indicate significance among treated cells calculated using one-way ANOVA and Tukey post-test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Differential function of ADAM10 in epithelial regeneration. A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr), as indicated in the graph legend. Cells were grown in 96-well plates until confluence and treated for 2 h with mitomycin (5 µg/mL) to avoid cell proliferation. In ( A , B ), cells were pre-incubated with 0.1% DMSO (vehicle control) or with ADAM10 inhibitor GI254023X (10 μM). Subsequently, cells were either left unstimulated or stimulated with ExoA (100 ng/mL). After 4 h, the stimulant was removed and the cells were investigated for wound closure after automated scratch induction over a period of 24 h using a live cell imaging system. In ( C , D ), the same setup was used. Additionally, GI254023X was added after ExoA treatment and scratch induction. Data are shown as a percentage of wound closure relative to control treated cells ( A , C representative images; B , D automated quantifications). Quantitative data are shown as means + SD of six independent experiments. Asterisks indicate significance among treated cells calculated using one-way ANOVA and Tukey post-test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Techniques: Transduction, shRNA, Incubation, Live Cell Imaging

$Functional implication of exosomal ADAM10 P. aeruginosa infection. ( A ) The 2 × 10 7 A549 cells were left unstimulated or infected with P. aeruginosa (MOI 5) in serum free medium for 2 h. Subsequently, the cell supernatant was subjected to differential centrifugation (300, 1000, 10,000, 100,000 g ). The pellets obtained in each sequential centrifugation step and the cells were lysed and subjected to Western blot analysis together with the unfractionated supernatants. Membranes were probed against ADAM10 (C-terminal antibody), Flotiline-1 and CD9 (positive markers for exosomes). ( B ) Extracellular vesicles from P. aeruginosa infected cells were collected as described in A and subjected to sucrose density gradient centrifugation after centrifugation at 100,000 g , Purity of fractions was controlled by optical density measurements, and fractions were subjected to Western blot analysis. Membranes were probed against ADAM10, Flotiline-1 and CD9. Notably, the ADAM10 positive fractions were identified as exosomes by the exosomal markers and the density. In ( A , B ), representative blots of at least three independent experiments are shown. ( C ) A549 cells were transfected with a plasmid encoding for AP-BTC and seeded at equal density. Cells were pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left untreated (no exosomes) or co-incubated with exosomes derived from either non-infected or P. aeruginosa infected (MOI 5 for 2) cells. After 2 h, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. ( D ) A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr) as indicated in the graph legend. Cells for activity measurement were transfected with a plasmid encoding for AP-BTC prior to seeding. Cells for exosome preparation were not transfected. Cells were either left untreated or co-incubated with exosomes prepared from P. aeruginosa infected (MOI 5 for 2 h) scramble or A10 KD cells, respectively. After 2 h, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using two-way ANOVA and Tukey post-test in ( C ) and one-way ANOVA and Tukey post-test in ( D ) (* p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. not significant).$

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Functional implication of exosomal ADAM10 P. aeruginosa infection. ( A ) The 2 × 10 7 A549 cells were left unstimulated or infected with P. aeruginosa (MOI 5) in serum free medium for 2 h. Subsequently, the cell supernatant was subjected to differential centrifugation (300, 1000, 10,000, 100,000 g ). The pellets obtained in each sequential centrifugation step and the cells were lysed and subjected to Western blot analysis together with the unfractionated supernatants. Membranes were probed against ADAM10 (C-terminal antibody), Flotiline-1 and CD9 (positive markers for exosomes). ( B ) Extracellular vesicles from P. aeruginosa infected cells were collected as described in A and subjected to sucrose density gradient centrifugation after centrifugation at 100,000 g , Purity of fractions was controlled by optical density measurements, and fractions were subjected to Western blot analysis. Membranes were probed against ADAM10, Flotiline-1 and CD9. Notably, the ADAM10 positive fractions were identified as exosomes by the exosomal markers and the density. In ( A , B ), representative blots of at least three independent experiments are shown. ( C ) A549 cells were transfected with a plasmid encoding for AP-BTC and seeded at equal density. Cells were pre-incubated with ADAM10 inhibitor GI254023X (10 μM) or 0.1% DMSO (vehicle control) for 30 min. Subsequently, cells were left untreated (no exosomes) or co-incubated with exosomes derived from either non-infected or P. aeruginosa infected (MOI 5 for 2) cells. After 2 h, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. ( D ) A549 cells were transduced with lentivirus encoding shRNA against ADAM10 for knockdown (KD) (A10-KD1 or 10-KD2) or an unspecific shRNA (scramble, scr) as indicated in the graph legend. Cells for activity measurement were transfected with a plasmid encoding for AP-BTC prior to seeding. Cells for exosome preparation were not transfected. Cells were either left untreated or co-incubated with exosomes prepared from P. aeruginosa infected (MOI 5 for 2 h) scramble or A10 KD cells, respectively. After 2 h, AP activity was determined in the cell lysate and supernatant to quantify the relative betacellulin cleavage and release. Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance among treated cells calculated using two-way ANOVA and Tukey post-test in ( C ) and one-way ANOVA and Tukey post-test in ( D ) (* p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. not significant).

Techniques: Functional Assay, Infection, Centrifugation, Western Blot, Gradient Centrifugation, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Activity Assay, Transduction, shRNA

Pathogen-specific activation of ADAM10 depends on the toxin repertoire and calcium increase. ( A ) A549 cells were grown to confluence and either left unstimulated or infected with heat-inactivated P. aeruginosa (multiplicity of infection of 5 (MOI 5). Samples were taken after an incubation time of 30, 60, 120 or 240 min. ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against GAPDH served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A ). ( B , C , D ) A549 cells were grown to 70% confluency on poly-L-lysine coated glass coverslips and either left unstimulated (PBS) or stimulated with ExoA (100 ng/mL) for 4 h in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C , D ). Subsequently, the cells loaded with 5 μM Fura-2 AM for 45 min at 37 °C followed by calcium signaling recording over 1000 s in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C ) or calcium free Tyrode’s solution followed by addition of 2 mM calcium after 120 s of calcium signaling recording ( D ). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference among treated cells at the indicated time point calculated using two-way ANOVA and Bonferroni post-test for F340/380 ratio over time and one-way ANOVA and Tukey post-test for area under the curve (* p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001, n.s. not significant). In ( A ), significance was analyzed by two tailed two samples t-test. No significant differences were observed.

Journal: International Journal of Molecular Sciences

Article Title: Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans

doi: 10.3390/ijms23031259

Figure Lengend Snippet: Pathogen-specific activation of ADAM10 depends on the toxin repertoire and calcium increase. ( A ) A549 cells were grown to confluence and either left unstimulated or infected with heat-inactivated P. aeruginosa (multiplicity of infection of 5 (MOI 5). Samples were taken after an incubation time of 30, 60, 120 or 240 min. ADAM10 protein expression and maturation was investigated in cell lysates by Western blot probing with an antibody against the C-terminus (intracellular part). Probing against GAPDH served as loading control. Band intensities of the pro-form (100 kDa) and the mature form (70 kDa) were quantified by densitometry and normalized to the expression of the unstimulated cells (0 h). A representative blot is shown in ( A ). ( B , C , D ) A549 cells were grown to 70% confluency on poly-L-lysine coated glass coverslips and either left unstimulated (PBS) or stimulated with ExoA (100 ng/mL) for 4 h in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C , D ). Subsequently, the cells loaded with 5 μM Fura-2 AM for 45 min at 37 °C followed by calcium signaling recording over 1000 s in Tyrode’s solution ( B ), calcium free Tyrode’s solution ( C ) or calcium free Tyrode’s solution followed by addition of 2 mM calcium after 120 s of calcium signaling recording ( D ). Quantitative data are shown as means + SD of three independent experiments. Asterisks indicate significance difference among treated cells at the indicated time point calculated using two-way ANOVA and Bonferroni post-test for F340/380 ratio over time and one-way ANOVA and Tukey post-test for area under the curve (* p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001, n.s. not significant). In ( A ), significance was analyzed by two tailed two samples t-test. No significant differences were observed.

Techniques: Activation Assay, Infection, Incubation, Expressing, Western Blot, Two Tailed Test

ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P < 0.05, n = 3–4. (D) Quantification of confrontation experiments at 48 hours using ephrin-A1–expressing cells (EFNA1) in contact with “unlike” control cells that had been treated with DMSO or the EGFR inhibitor, AG1478 (AG). Cells were either pretreated before the initiation of confrontation for 1 hour (AG pretreat) or treated with inhibitor 5 (AG @ 5 hrs) or 24 hours (AG @ 24 hrs) after initiation of confrontation. (E, F) Various concentrations of EGF (0.1, 1, 10, or 100 ng/mL) were added to the culture medium of these ephrin-A1 and control cell cocultures after pretreatment with GIX for 5 hours. Images (E) and quantification (F) are shown 48 hours after confrontation. Solid white lines mark the midline where the silicone divider was present at the time of its removal. White dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 3–4.

Journal: Investigative Ophthalmology & Visual Science

Article Title: EphA2/Ephrin-A1 Mediate Corneal Epithelial Cell Compartmentalization via ADAM10 Regulation of EGFR Signaling

doi: 10.1167/iovs.17-22941

Figure Lengend Snippet: ADAM10 mediates Ephrin-A1/EphA2 boundary organization via EGFR signaling. (A) E-cadherin (E-cad; top) and ADAM10 (bottom) immunofluorescence staining in human anterior segmental epithelium. Scale bar denotes 100 μm. (B) E-cadherin staining of control cells (Control, green) confronted by “like” control cells (Control, red) or ephrin-A1–expressing cells (EFNA1, red) confronted by “unlike” control cells (Control, green; bottom) in the presence of general MMP inhibitor, TAPI, or a specific ADAM10 inhibitor, GI254023X (GIX). Red dotted lines indicate the boundary between the two cell populations 48 hours after initiation of confrontation. Scale bar denotes 80 μm. (C) Quantification of confrontation experiments at 48 hours in cocultures treated with DMSO, GIX, LY294002 (LY), Y-27632 (Y), or U0126 (U). * P < 0.05, n = 3–4. (D) Quantification of confrontation experiments at 48 hours using ephrin-A1–expressing cells (EFNA1) in contact with “unlike” control cells that had been treated with DMSO or the EGFR inhibitor, AG1478 (AG). Cells were either pretreated before the initiation of confrontation for 1 hour (AG pretreat) or treated with inhibitor 5 (AG @ 5 hrs) or 24 hours (AG @ 24 hrs) after initiation of confrontation. (E, F) Various concentrations of EGF (0.1, 1, 10, or 100 ng/mL) were added to the culture medium of these ephrin-A1 and control cell cocultures after pretreatment with GIX for 5 hours. Images (E) and quantification (F) are shown 48 hours after confrontation. Solid white lines mark the midline where the silicone divider was present at the time of its removal. White dotted lines indicate the boundary between the two different cell populations 48 hours after initiation of confrontation. n = 3–4.

Article Snippet: Frozen sections (5 μm) of optimal cutting temperature compound (OCT)-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% donkey serum in PBS, and incubated overnight with the following primary antibodies: a goat anti-human EphA2 (AF3035) or a goat anti-mouse EphA2 (AF639; R&D Systems), a rabbit anti-ephrin-A1 (V18), a rabbit polyclonal antibody against ADAM10 (Abcam), or a mouse monoclonal antibody against E-cadherin (HECD1; Abcam).

Techniques: Immunofluorescence, Staining, Control, Expressing

(A) Thioflavin-S staining. (B) 6-E10 immunohistochemical staining. Neither Thioflavin-S-positive fibrillary plaques nor 6E10-immunopositive diffuse plaques were observed in the hippocampus of the both groups of mice. (C) Representative immunoblot and (D-E) corresponding densitometry analysis for soluble amyloid precursor protein-α peptides (sAPPα) and soluble Aβ peptides. (F) Representative immunoblot and (G) corresponding densitometry analysis for APP secretases, including a-disintegrin and metalloproteinase 10 (ADAM10), (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and presenilin1 (PS1), and Aβ-degrading enzymes including neprilysin (NEP) and insulin-degrading enzyme (IDE). Data represent means ± SEM. Two-way ANOVA followed by post-hoc multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001, compared to Control; #P < 0.05; ###P < 0.001 compared to WT. n = 4 in each group.

Journal: bioRxiv

Article Title: Anxiety-like but not despair-like behaviors are further aggravated by chronic mild stress in the early stages of APP swe /PS1dE9 transgenic mice

doi: 10.1101/202283

Figure Lengend Snippet: (A) Thioflavin-S staining. (B) 6-E10 immunohistochemical staining. Neither Thioflavin-S-positive fibrillary plaques nor 6E10-immunopositive diffuse plaques were observed in the hippocampus of the both groups of mice. (C) Representative immunoblot and (D-E) corresponding densitometry analysis for soluble amyloid precursor protein-α peptides (sAPPα) and soluble Aβ peptides. (F) Representative immunoblot and (G) corresponding densitometry analysis for APP secretases, including a-disintegrin and metalloproteinase 10 (ADAM10), (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and presenilin1 (PS1), and Aβ-degrading enzymes including neprilysin (NEP) and insulin-degrading enzyme (IDE). Data represent means ± SEM. Two-way ANOVA followed by post-hoc multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001, compared to Control; #P < 0.05; ###P < 0.001 compared to WT. n = 4 in each group.

Article Snippet: After being blocked with 5% milk, the bands were incubated with rabbit polyclonal antibody against ADAM10 (1:1000; Millipore), ASC (1:1500; Santa Cruz), BDNF (1:300; Abcam), CREB (1:1000; Cell Signaling Technology), Caspase1 (1:1000; Millipore), IDE (1:800; Abcam), IL-1β (1:1000; Millipore), IL-6 (1:1000; Abcam), NEP (1:800; Millipore), PS1 (1:1000; Sigma), NLRP3 (1:1000; AdipoGen), p-CREB (1:1000; Cell Signaling Technology), procaspase 1 (1:500; Millipore), sAPPα (1:800; IBL), TrkB (1:500; Santa Cruz), TNF-α (1:1000; Abcam), rabbit monoclonal antibody against Aβ 1-42 (1:1000; Abcam), or mouse monoclonal antibody against BACE1 (1:1000; Millipore) at 4°C overnight.

Techniques: Staining, Immunohistochemical staining, Western Blot, Comparison, Control

(A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) U87 CAR cells plated on poly-L-lysine coated plates were pre-incubated for 45 minutes with a variety of protease inhibitors (10 µM pepstatin A, 10 µM leupeptin, 10 µM E64, 250 µM O-phenanthroline, 25 µM TAPI-1) followed by 3 hours of treatment with 1 µM of PMA. None of the treatments were toxic to the cells under these conditions and concentrations of inhibitors. CAR ECD released into conditioned media was detected via Western blot using anti-CAR N-term. antibody 2240. The broad-spectrum metalloprotease inhibitors TAPI-1 and O-phenathroline decreased PMA-stimulated CAR ECD shedding, while the aspartyl protease inhibitor pepstatin, the cysteine protease inhibitor E64, and the cysteine/serine protease inhibitor leupeptin had no effect. Also shown are Western blots of full-length CAR from the corresponding cell lysates (anti-CAR C-term. antibody RP291). (B) U87 CAR cells were treated with PMA (1 µM) or DMSO vehicle, in the presence of 25 µM of the broad spectrum metalloprotease inhibitor GM6001 or its negative control. GM6001, but not its negative control, inhibited PMA-stimulated shedding of CAR ECD. (C) U87 CAR cells were incubated for 3 hours with 1 µM PMA along with 10 µg/ml of TIMPs 1, 2 or 3. TIMP1 and TIMP3, but not TIMP2, decreased PMA-mediated ECD shedding of CAR, suggesting that ADAM10 may be a sheddase. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: Incubation, Western Blot, Protease Inhibitor, Negative Control

(A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) U251N cells stably expressing CAR were transfected with empty plasmid (mock) or ADAM10 plasmid. 24 hours after transfection, cells were washed and incubated in opti-MEM for 24 hours, and conditioned media and cell lysates were analyzed by Western blots using anti-CAR N-terminus antibody (2239). Overexpression of ADAM10 increased constitutive CAR ECD shedding. (B) U87 CAR cells were treated with 10 μM purified ADAM10 prodomain (versus an equivalent volume of buffer as a control), and conditioned media and cell lysates were collected as previously described. A Western blot for CAR extracellular domain (2240 antibody) shows that the prodomain of ADAM10, which inhibits ADAM10 activity, decreased CAR ECD shedding.

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Over Expression, Purification, Control, Activity Assay

(A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) A Western blot for ADAM10 using cell lysates of U87 CAR cells containing either control shRNA or ADAM10 (#6676) shRNA, in biological duplicates. Equal amounts of proteins were loaded on SDS-PAGE gel. Anti-GAPDH antibody was used as a loading control. Quantification of mean ADAM10 band intensities normalized over GAPDH revealed a decrease of approximately 60% with anti-ADAM10 shRNA compared to control shRNA. (B) A constitutive shedding experiment was performed using U87 CAR stable cell lines containing either control shRNA or anti-ADAM10 shRNA (#6676). Conditioned media and cell lysates were collected after 24 hours of incubation of cells in opti-MEM, and Western blotting was done using the anti-CAR N-terminus antibody 2239. With anti-ADAM10 shRNA, there was a significant decrease (40%) in the levels of shed CAR. Results from 4 independent experiments performed in duplicates were quantified (unpaired t-test; p=0.0004 (***)). (C) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6676) were treated with 1 µM PMA. Conditioned media and cell lysates were collected after 3 hours, and Western blots were performed using the anti-CAR N-terminus antibody 2239. With shRNA knockdown of ADAM10, there was a significant decrease of 41% in levels of shed CAR compared to control shRNA. Results from 3 independent experiments performed in duplicates were quantified (one-way ANOVA with Tukey’s multiple comparison test; *** = p < 0.001). (D) U87 CAR cells containing either control shRNA or ADAM10 shRNA (#6675) were treated with 1.5 µM ionomycin (vs. DMSO vehicle). Conditioned media and cell lysates were collected after 30 minutes of treatment, and Western blots were performed using the anti-CAR N-terminus antibody 2240. With shRNA knockdown of ADAM10, there was a significant decrease of 52% in levels of shed CAR with ionomycin treatment compared to control shRNA. Results from 3 independent experiments (n=3 per group) were quantified (one-way ANOVA with Bonferroni’s multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: Western Blot, Control, shRNA, SDS Page, Stable Transfection, Incubation, Knockdown, Comparison

(A) shRNA stable cell lines of U251N CAR cells were transfected with either empty plasmid (mock) or an shRNA-resistant construct of ADAM10. 18-24 hours post-transfection, cells were washed and incubated in opti-MEM. Conditioned media and cell lysates were collected 24 hours later and analyzed by Western blot for CAR extracellular domain (anti-CAR antibody 2239). The shRNA-resistant ADAM10 mutant partially rescued CAR shedding in the ADAM10 (6676) shRNA cell line, compared to transfecting this cell line with empty plasmid (third and fourth Western blot bands from the left). (B) The band intensities of shed CAR detected by Western blot were quantified from 4 independent experiments (One-way ANOVA with Newman-Keuls multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) shRNA stable cell lines of U251N CAR cells were transfected with either empty plasmid (mock) or an shRNA-resistant construct of ADAM10. 18-24 hours post-transfection, cells were washed and incubated in opti-MEM. Conditioned media and cell lysates were collected 24 hours later and analyzed by Western blot for CAR extracellular domain (anti-CAR antibody 2239). The shRNA-resistant ADAM10 mutant partially rescued CAR shedding in the ADAM10 (6676) shRNA cell line, compared to transfecting this cell line with empty plasmid (third and fourth Western blot bands from the left). (B) The band intensities of shed CAR detected by Western blot were quantified from 4 independent experiments (One-way ANOVA with Newman-Keuls multiple comparison test; * = p < 0.05, ** = p < 0.01, *** = p < 0.001).

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: shRNA, Stable Transfection, Transfection, Plasmid Preparation, Construct, Incubation, Western Blot, Mutagenesis, Comparison

(A) A schematic showing putative ADAM10 cleavage sites on CAR’s extracellular domain (arrows), as obtained from in vitro peptide digestion and mass spectrometry (Figure S4). (B) HEK 293 stable cell populations were generated to express wild-type CAR, the mutant MLRLAAAA or the mutant Δ221-232. The MLRLAAAA mutant did not shed into conditioned media of HEK 293 cells, while the Δ221-232 mutant shed its ECD similarly to wild-type CAR. Cell surface biotinylation experiments revealed that wild-type CAR and the two mutants are expressed similarly on the cell surface. Note that the low level of endogenous CAR in HEK 293 was detected after enrichment of cell surface biotinylated proteins (mock lane). Western blotting was performed using the anti-CAR N-terminus antibody 2240.

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) A schematic showing putative ADAM10 cleavage sites on CAR’s extracellular domain (arrows), as obtained from in vitro peptide digestion and mass spectrometry (Figure S4). (B) HEK 293 stable cell populations were generated to express wild-type CAR, the mutant MLRLAAAA or the mutant Δ221-232. The MLRLAAAA mutant did not shed into conditioned media of HEK 293 cells, while the Δ221-232 mutant shed its ECD similarly to wild-type CAR. Cell surface biotinylation experiments revealed that wild-type CAR and the two mutants are expressed similarly on the cell surface. Note that the low level of endogenous CAR in HEK 293 was detected after enrichment of cell surface biotinylated proteins (mock lane). Western blotting was performed using the anti-CAR N-terminus antibody 2240.

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: In Vitro, Mass Spectrometry, Stable Transfection, Generated, Mutagenesis, Western Blot

(A) A Western blot for CAR’s intracellular domain (antibody RP291) shows lower molecular weight fragments in U87 CAR lysates at approximately 20 kDa and 14 kDa (CTF1 and CTF2, respectively). Full-length CAR (CAR FL) migrates at 50 kDa. (B) U87 CAR cells were treated with the γ-secretase inhibitors MG132 or DAPT for 16 hours. Equal amounts of proteins from cell lysates were analyzed by Western blot using the anti-CAR intracellular domain antibody RP291. Drug treatments resulted in accumulation of CAR CTF1 (20 kDa) and a decrease in CAR CTF2 (14 kDa) levels in a dose-dependent manner. (C) Overnight treatment of U87 CAR cells with Compound E, another inhibitor of the γ-secretase complex, at the indicated concentrations also diminished RIP of CAR. Equal quantities of proteins from cell lysates were analyzed by SDS-PAGE and Western blot using the anti-CAR intracellular domain antibody RP291. With increasing concentrations of Compound E, there was a corresponding accumulation of a 20 kDa fragment (CAR CTF1) and a decrease in a 14 kDa fragment (CAR CTF2). 3 different exposure times of the Western blot are shown. (D) A model of CAR proteolysis, with molecular weights of the resulting fragments indicated. Cleavage of CAR by ADAM10 (represented by the top pair of scissors) releases a 32 kDa fragment (CAR ECD) into the extracellular environment. The remaining 20 kDa fragment (CTF1) is processed by the γ-secretase complex (represented by the lower pair of scissors), generating a 14 kDa fragment (CTF2).

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) A Western blot for CAR’s intracellular domain (antibody RP291) shows lower molecular weight fragments in U87 CAR lysates at approximately 20 kDa and 14 kDa (CTF1 and CTF2, respectively). Full-length CAR (CAR FL) migrates at 50 kDa. (B) U87 CAR cells were treated with the γ-secretase inhibitors MG132 or DAPT for 16 hours. Equal amounts of proteins from cell lysates were analyzed by Western blot using the anti-CAR intracellular domain antibody RP291. Drug treatments resulted in accumulation of CAR CTF1 (20 kDa) and a decrease in CAR CTF2 (14 kDa) levels in a dose-dependent manner. (C) Overnight treatment of U87 CAR cells with Compound E, another inhibitor of the γ-secretase complex, at the indicated concentrations also diminished RIP of CAR. Equal quantities of proteins from cell lysates were analyzed by SDS-PAGE and Western blot using the anti-CAR intracellular domain antibody RP291. With increasing concentrations of Compound E, there was a corresponding accumulation of a 20 kDa fragment (CAR CTF1) and a decrease in a 14 kDa fragment (CAR CTF2). 3 different exposure times of the Western blot are shown. (D) A model of CAR proteolysis, with molecular weights of the resulting fragments indicated. Cleavage of CAR by ADAM10 (represented by the top pair of scissors) releases a 32 kDa fragment (CAR ECD) into the extracellular environment. The remaining 20 kDa fragment (CTF1) is processed by the γ-secretase complex (represented by the lower pair of scissors), generating a 14 kDa fragment (CTF2).

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: Western Blot, Molecular Weight, SDS Page

(A) Stable cell lines of U87-MG and U251N expressing either the V5 tag alone (mock) or CAR with a C-terminal V5 tag were generated. Equal amounts of cell lysates were analyzed by SDS-PAGE and Western blot using a mouse monoclonal antibody raised against the V5 tag. Full-length CAR, CTF1 and CTF2 were detected similarly to lysates of U87 CAR cells probed with anti-CAR C-term. antibody RP291 (Figure 8A). (B) U251N V5 and U251N CAR-V5 cells were treated overnight with MG132 (25 µM) or DMSO vehicle control. Equal amounts of proteins from cell lysates were used for anti-V5 Western blots. In the case of the MG132-treated cells, CTF1 levels accumulated while CTF2 nearly disappeared, similar to previous experiments with the U87 CAR cell line (Figure 8B). (C) MEF wild-type (MEF WT) or PS 1- and 2-knockout MEF cells (MEF PS1/2 KO) were infected with lentivirus to express full-length CAR with a C-terminal V5 tag. Cells were lysed 3 days post-infection and lysates were analyzed by Western blot using antibody raised against the V5 tag. MEF WT cells, but not MEF PS1/2 KO cells, contained CAR CTF2, indicating that presenilin is required for generation of the 14 kDa CTF2 fragment of CAR. (D) Verification of knockdown in ADAM10 expression in U87 CAR-V5 cells using ADAM10 shRNA (#6675); shown are anti-ADAM10 and anti-GAPDH Western blots. Lysates were also analyzed by Western blot using antibody raised against the V5 tag. The ADAM10 shRNA stable cell line had a decreased CAR CTF1 level, as expected. CAR CTF2 levels also decreased, indicating that shedding is a prerequisite for RIP of CAR.

Journal: PLoS ONE

Article Title: The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

doi: 10.1371/journal.pone.0073296

Figure Lengend Snippet: (A) Stable cell lines of U87-MG and U251N expressing either the V5 tag alone (mock) or CAR with a C-terminal V5 tag were generated. Equal amounts of cell lysates were analyzed by SDS-PAGE and Western blot using a mouse monoclonal antibody raised against the V5 tag. Full-length CAR, CTF1 and CTF2 were detected similarly to lysates of U87 CAR cells probed with anti-CAR C-term. antibody RP291 (Figure 8A). (B) U251N V5 and U251N CAR-V5 cells were treated overnight with MG132 (25 µM) or DMSO vehicle control. Equal amounts of proteins from cell lysates were used for anti-V5 Western blots. In the case of the MG132-treated cells, CTF1 levels accumulated while CTF2 nearly disappeared, similar to previous experiments with the U87 CAR cell line (Figure 8B). (C) MEF wild-type (MEF WT) or PS 1- and 2-knockout MEF cells (MEF PS1/2 KO) were infected with lentivirus to express full-length CAR with a C-terminal V5 tag. Cells were lysed 3 days post-infection and lysates were analyzed by Western blot using antibody raised against the V5 tag. MEF WT cells, but not MEF PS1/2 KO cells, contained CAR CTF2, indicating that presenilin is required for generation of the 14 kDa CTF2 fragment of CAR. (D) Verification of knockdown in ADAM10 expression in U87 CAR-V5 cells using ADAM10 shRNA (#6675); shown are anti-ADAM10 and anti-GAPDH Western blots. Lysates were also analyzed by Western blot using antibody raised against the V5 tag. The ADAM10 shRNA stable cell line had a decreased CAR CTF1 level, as expected. CAR CTF2 levels also decreased, indicating that shedding is a prerequisite for RIP of CAR.

Article Snippet: Rabbit polyclonal antibody raised against ADAM10 was from AnaSpec, Inc.

Techniques: Stable Transfection, Expressing, Generated, SDS Page, Western Blot, Control, Knock-Out, Infection, Knockdown, shRNA

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rabbit polyclonal antibody against α-disintegrin and metalloproteinase 10 (adam10) (Millipore)

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