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Millipore rabbit antibodies against adam10

Protein levels of APP and <t>ADAM10</t> were decreased in diabetic brain microvessels. (a–e) Mouse cerebral microvessels were isolated from whole brain of the mice exposed to two weeks or two months of hyperglycemia. Western blot was carried out for detection of (a) APP (two weeks, n = 5; two months, n = 9), (b) ADAM10 (two weeks, n = 5; two months, n = 9), (c) ADAM17 (two weeks, n = 5, two months n = 7), (d) ADAM9 (two weeks, n = 5; two months, n = 7), and (e) BACE1 (two weeks, n = 5, two months, n = 7). *P < 0.05, compared to control mice. (f) APP protein expression was significantly reduced in hippocampal microvessels of mice two weeks after developing hyperglycemia (n = 6, *P < 0.05). (g) ADAM10 protein levels were not significantly changed in hippocampal microvessels of two week-T1D mice (n = 6). Cropped blots are displayed.

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1) Product Images from "Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice"

Article Title: Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1177/0271678X17746981

Protein levels of APP and ADAM10 were decreased in diabetic brain microvessels. (a–e) Mouse cerebral microvessels were isolated from whole brain of the mice exposed to two weeks or two months of hyperglycemia. Western blot was carried out for detection of (a) APP (two weeks, n = 5; two months, n = 9), (b) ADAM10 (two weeks, n = 5; two months, n = 9), (c) ADAM17 (two weeks, n = 5, two months n = 7), (d) ADAM9 (two weeks, n = 5; two months, n = 7), and (e) BACE1 (two weeks, n = 5, two months, n = 7). *P < 0.05, compared to control mice. (f) APP protein expression was significantly reduced in hippocampal microvessels of mice two weeks after developing hyperglycemia (n = 6, *P < 0.05). (g) ADAM10 protein levels were not significantly changed in hippocampal microvessels of two week-T1D mice (n = 6). Cropped blots are displayed.

Figure Legend Snippet: Protein levels of APP and ADAM10 were decreased in diabetic brain microvessels. (a–e) Mouse cerebral microvessels were isolated from whole brain of the mice exposed to two weeks or two months of hyperglycemia. Western blot was carried out for detection of (a) APP (two weeks, n = 5; two months, n = 9), (b) ADAM10 (two weeks, n = 5; two months, n = 9), (c) ADAM17 (two weeks, n = 5, two months n = 7), (d) ADAM9 (two weeks, n = 5; two months, n = 7), and (e) BACE1 (two weeks, n = 5, two months, n = 7). *P < 0.05, compared to control mice. (f) APP protein expression was significantly reduced in hippocampal microvessels of mice two weeks after developing hyperglycemia (n = 6, *P < 0.05). (g) ADAM10 protein levels were not significantly changed in hippocampal microvessels of two week-T1D mice (n = 6). Cropped blots are displayed.

Techniques Used: Isolation, Western Blot, Expressing

Knockdown of AC3 suppressed ADAM10 protein levels in human BMECs. (a) AC3 mRNA levels were significantly reduced in human BMECs treated with AC3-siRNA for three days (n = 5, *P < 0.05, compare to cells treated with Ct-siRNA). (b) AC3-siRNA treatment for three days suppressed ADAM10 protein expression (n = 8,*P < 0.05, compared to cells treated with Ct-siRNA). AC3-siRNA treatment did not affect expression of APP (c, n = 8) or BACE1 (d, n = 3).

Figure Legend Snippet: Knockdown of AC3 suppressed ADAM10 protein levels in human BMECs. (a) AC3 mRNA levels were significantly reduced in human BMECs treated with AC3-siRNA for three days (n = 5, *P < 0.05, compare to cells treated with Ct-siRNA). (b) AC3-siRNA treatment for three days suppressed ADAM10 protein expression (n = 8,*P < 0.05, compared to cells treated with Ct-siRNA). AC3-siRNA treatment did not affect expression of APP (c, n = 8) or BACE1 (d, n = 3).

Techniques Used: Expressing

In cerebral microvessels of T1D mice, reduced expression of ADAM10 and APP caused by decreased AC3 signaling and increased production of TXA2, respectively, may contribute to low levels of sAPPα in the hippocampus. We speculate that the reduced sAPPα levels in the hippocampus may be one of mechanisms responsible for cognitive dysfunction.

Figure Legend Snippet: In cerebral microvessels of T1D mice, reduced expression of ADAM10 and APP caused by decreased AC3 signaling and increased production of TXA2, respectively, may contribute to low levels of sAPPα in the hippocampus. We speculate that the reduced sAPPα levels in the hippocampus may be one of mechanisms responsible for cognitive dysfunction.

Techniques Used: Expressing

Isolation:

Article Title: Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice
Article Snippet: Rabbit antibodies against ADAM10 and ADAM17 were obtained from EMD Millipore Corporation (Temecula, CA).

Western Blot:

Expressing:

Image Search Results

Figure 1. Histological analysis of primary RB tumors. ADAM17 (a) and ADAM10 (b) expression in exemplary parafﬁn sections of human RB patient tumors. Immunohistochemistry was detected by diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 600 µM and 200 µM (magniﬁed insets).

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 1. Histological analysis of primary RB tumors. ADAM17 (a) and ADAM10 (b) expression in exemplary parafﬁn sections of human RB patient tumors. Immunohistochemistry was detected by diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 600 µM and 200 µM (magniﬁed insets).

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Expressing, Immunohistochemistry, Staining

Figure 2. Veriﬁcation of ADAM10 and ADAM17 knockdown efﬁciency. Efﬁcient stable, lentiviral ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) double knock- down was veriﬁed by quantitative real-time PCR (a,d) and western blot analyses in Y79 (b,c) and WERI-Rb1 cells (e,f). Indicated intensity ratios relative to ß-actin, used as a loading control, were cal- culated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 2. Veriﬁcation of ADAM10 and ADAM17 knockdown efﬁciency. Efﬁcient stable, lentiviral ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) double knock- down was veriﬁed by quantitative real-time PCR (a,d) and western blot analyses in Y79 (b,c) and WERI-Rb1 cells (e,f). Indicated intensity ratios relative to ß-actin, used as a loading control, were cal- culated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Figure 3. Effects of ADAM10/17 single and double knockdown on cell viability and proliferation of RB cells. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases cell viability and proliferation levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by WST-1 assays (a), and BrdU stains (b,c). Values are means of three independent experiments ± SEM. ns p > 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 3. Effects of ADAM10/17 single and double knockdown on cell viability and proliferation of RB cells. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases cell viability and proliferation levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by WST-1 assays (a), and BrdU stains (b,c). Values are means of three independent experiments ± SEM. ns p > 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Techniques: Knockdown, Control

Figure 4. Effects of ADAM10/17 single and double knockdown on cell growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases growth of Y79 (a,c,e) and WERI-Rb1 cells (b,d,f) compared to control cells (ctr) as revealed by growth curve analysis. Values are means of three independent experiments ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 4. Effects of ADAM10/17 single and double knockdown on cell growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases growth of Y79 (a,c,e) and WERI-Rb1 cells (b,d,f) compared to control cells (ctr) as revealed by growth curve analysis. Values are means of three independent experiments ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Techniques: Knockdown, Control

Figure 5. Effects of ADAM10/17 single and double knockdown on apoptosis and anchorage in- dependent growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown increases caspase mediated apoptosis levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by DAPI cell counts (a) and caspase3/7 assays (b). Both RB cell lines show signiﬁcantly reduced colony formation capacity (c) and colony sizes (d,e) after ADAM10 and ADAM 17 single or double knockdown as revealed by soft agarose assays. Values are means of three independent experiments ± SEM. ns > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 5. Effects of ADAM10/17 single and double knockdown on apoptosis and anchorage in- dependent growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown increases caspase mediated apoptosis levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by DAPI cell counts (a) and caspase3/7 assays (b). Both RB cell lines show signiﬁcantly reduced colony formation capacity (c) and colony sizes (d,e) after ADAM10 and ADAM 17 single or double knockdown as revealed by soft agarose assays. Values are means of three independent experiments ± SEM. ns > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Techniques: Knockdown, Control

Figure 6. Effect of lentiviral ADAM10/17 single and double knockdown on tumor formation of RB cell lines in vivo as revealed by chick chorioallantoic membrane (CAM) assays. Photographs of CAM tumors in situ (upper row) and ruler measurements (in cm) of excised CAM tumors (lower row) 7 days after inoculating the Y79 (a) and WERI-Rb1 (b) RB cell onto the CAM. Quantiﬁcation of weight (c) and size (d) of CAM tumors developing from ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) depleted Y79 and WERI-Rb1 cell lines in comparison the control cells (ctr). Values are means of at least three independent experiments ± SEM. ns p > 0.05; * p < 0.05; *** p < 0.001; and **** p < 0.0001 statistical differences compared to the control group calculated by Student’s t-test. (e) GFP antibody staining of stably GFP expressing Y79 and WERI-Rb1 cells in CAM tumor parafﬁn sections. Immunohistochemistry was revealed using diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 2 mm and 300 µM (magniﬁed insets). #: blood vessel in CAM mesoderm, *: GFP-positive (GFP+) RB tumor cells, arrowheads: solid GFP-positive RB tumor mass.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 6. Effect of lentiviral ADAM10/17 single and double knockdown on tumor formation of RB cell lines in vivo as revealed by chick chorioallantoic membrane (CAM) assays. Photographs of CAM tumors in situ (upper row) and ruler measurements (in cm) of excised CAM tumors (lower row) 7 days after inoculating the Y79 (a) and WERI-Rb1 (b) RB cell onto the CAM. Quantiﬁcation of weight (c) and size (d) of CAM tumors developing from ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) depleted Y79 and WERI-Rb1 cell lines in comparison the control cells (ctr). Values are means of at least three independent experiments ± SEM. ns p > 0.05; * p < 0.05; *** p < 0.001; and **** p < 0.0001 statistical differences compared to the control group calculated by Student’s t-test. (e) GFP antibody staining of stably GFP expressing Y79 and WERI-Rb1 cells in CAM tumor parafﬁn sections. Immunohistochemistry was revealed using diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 2 mm and 300 µM (magniﬁed insets). #: blood vessel in CAM mesoderm, *: GFP-positive (GFP+) RB tumor cells, arrowheads: solid GFP-positive RB tumor mass.

Techniques: Knockdown, In Vivo, Membrane, In Situ, Comparison, Control, Staining, Stable Transfection, Expressing, Immunohistochemistry

Figure 7. Effects of stable, lentiviral ADAM10/17 single or double knockdown on RB cell migration in vivo. (a) Desmin stains of blood vessels (red) in representative CAM whole mounts showing

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 7. Effects of stable, lentiviral ADAM10/17 single or double knockdown on RB cell migration in vivo. (a) Desmin stains of blood vessels (red) in representative CAM whole mounts showing

Techniques: Knockdown, Migration, In Vivo

Figure 8. Analysis of L1CAM ectodomain shedding after ADAM10/17 single and double knockdown in Y79 and WERI-Rb1 cell lines. Western blot analysis of ADAM mediated L1CAM ectodomain (L1– 200) shedding in cell culture supernatant of Y79 and WERI-Rb1 cells 72 h after ADAM10 (shADAM10), ADAM17 (shADAM17) single and ADAM10/17 (shADAM10/17) double knockdown in comparison to control cells (ctr). Representative western blots of L1 shedding upon ADAM knockdown (a) and quantiﬁcation of the L1CAM ectodomain expression reveals a signiﬁcant reduction of L1-200 shed- ding after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1 (b). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. * p < 0.05 and ** p < 0.01 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 8. Analysis of L1CAM ectodomain shedding after ADAM10/17 single and double knockdown in Y79 and WERI-Rb1 cell lines. Western blot analysis of ADAM mediated L1CAM ectodomain (L1– 200) shedding in cell culture supernatant of Y79 and WERI-Rb1 cells 72 h after ADAM10 (shADAM10), ADAM17 (shADAM17) single and ADAM10/17 (shADAM10/17) double knockdown in comparison to control cells (ctr). Representative western blots of L1 shedding upon ADAM knockdown (a) and quantiﬁcation of the L1CAM ectodomain expression reveals a signiﬁcant reduction of L1-200 shed- ding after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1 (b). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. * p < 0.05 and ** p < 0.01 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Techniques: Knockdown, Western Blot, Cell Culture, Comparison, Control, Expressing, Software

Figure 9. AKT and phospho-AKT (pAKT) expression levels after shRNA-mediated ADAM10/17 single (shADAM10 and shADAM17) or double knockdown (shADAM10/17) in the RB cell lines Y79 and WERI-Rb1 as revealed by western blot analysis. Representative western blots showing AKT and p-AKT expression levels (a,e) and quantiﬁcation of the p-AKT/AKT ratio (b–d,f–h) after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1. Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. ns p > 0.05 and * p < 0.05 statistical differences compared to the control group calculated by paired Student’s t-test.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 9. AKT and phospho-AKT (pAKT) expression levels after shRNA-mediated ADAM10/17 single (shADAM10 and shADAM17) or double knockdown (shADAM10/17) in the RB cell lines Y79 and WERI-Rb1 as revealed by western blot analysis. Representative western blots showing AKT and p-AKT expression levels (a,e) and quantiﬁcation of the p-AKT/AKT ratio (b–d,f–h) after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1. Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. ns p > 0.05 and * p < 0.05 statistical differences compared to the control group calculated by paired Student’s t-test.

Techniques: Expressing, shRNA, Knockdown, Western Blot, Control, Software

Figure 11. Correlation of miRNA-145, miRNA-152 and miRNA-365 expression with ADAM10 (a–c) and ADAM17 (d–f) expression levels in 15 individual RB patients as revealed by real-time PCR.

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 11. Correlation of miRNA-145, miRNA-152 and miRNA-365 expression with ADAM10 (a–c) and ADAM17 (d–f) expression levels in 15 individual RB patients as revealed by real-time PCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction

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

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice

doi: 10.1177/0271678X17746981

Figure Lengend Snippet: Protein levels of APP and ADAM10 were decreased in diabetic brain microvessels. (a–e) Mouse cerebral microvessels were isolated from whole brain of the mice exposed to two weeks or two months of hyperglycemia. Western blot was carried out for detection of (a) APP (two weeks, n = 5; two months, n = 9), (b) ADAM10 (two weeks, n = 5; two months, n = 9), (c) ADAM17 (two weeks, n = 5, two months n = 7), (d) ADAM9 (two weeks, n = 5; two months, n = 7), and (e) BACE1 (two weeks, n = 5, two months, n = 7). *P < 0.05, compared to control mice. (f) APP protein expression was significantly reduced in hippocampal microvessels of mice two weeks after developing hyperglycemia (n = 6, *P < 0.05). (g) ADAM10 protein levels were not significantly changed in hippocampal microvessels of two week-T1D mice (n = 6). Cropped blots are displayed.

Article Snippet: Rabbit antibodies against ADAM10 and ADAM17 were obtained from EMD Millipore Corporation (Temecula, CA).

Techniques: Isolation, Western Blot, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice

doi: 10.1177/0271678X17746981

Figure Lengend Snippet: Knockdown of AC3 suppressed ADAM10 protein levels in human BMECs. (a) AC3 mRNA levels were significantly reduced in human BMECs treated with AC3-siRNA for three days (n = 5, *P < 0.05, compare to cells treated with Ct-siRNA). (b) AC3-siRNA treatment for three days suppressed ADAM10 protein expression (n = 8,*P < 0.05, compared to cells treated with Ct-siRNA). AC3-siRNA treatment did not affect expression of APP (c, n = 8) or BACE1 (d, n = 3).

Article Snippet: Rabbit antibodies against ADAM10 and ADAM17 were obtained from EMD Millipore Corporation (Temecula, CA).

Techniques: Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice

doi: 10.1177/0271678X17746981

Figure Lengend Snippet: In cerebral microvessels of T1D mice, reduced expression of ADAM10 and APP caused by decreased AC3 signaling and increased production of TXA2, respectively, may contribute to low levels of sAPPα in the hippocampus. We speculate that the reduced sAPPα levels in the hippocampus may be one of mechanisms responsible for cognitive dysfunction.

Article Snippet: Rabbit antibodies against ADAM10 and ADAM17 were obtained from EMD Millipore Corporation (Temecula, CA).

Techniques: Expressing

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. ADAM10 expression of PANC-1 and MIA PACA-2 cells was determined when 2 μmol/l gemcitabine was added into cell culture. B. Cells were transfected with ADAM10 siRNA or control siRNA for 48 h and the expression of mRNA and protein of ADAM10 was evaluated by Q-PCR and western blot. C. sULBP2 in the culture supernatant was evaluated by ELISA. *P<0.05.

Journal: Oncotarget

Article Title: Gemcitabine inhibits immune escape of pancreatic cancer by down regulating the soluble ULBP2 protein

doi: 10.18632/oncotarget.11780

Figure Lengend Snippet: A. ADAM10 expression of PANC-1 and MIA PACA-2 cells was determined when 2 μmol/l gemcitabine was added into cell culture. B. Cells were transfected with ADAM10 siRNA or control siRNA for 48 h and the expression of mRNA and protein of ADAM10 was evaluated by Q-PCR and western blot. C. sULBP2 in the culture supernatant was evaluated by ELISA. *P<0.05.

Article Snippet: After being blocked with 5% nonfat milk, membranes were incubated overnight at 4 °C with rabbit primary antibodies against human ADAM10 (Boster, China) and human actin (Zhongshan Golden Bridge Biotechnology, China).

Techniques: Expressing, Cell Culture, Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay

The ADAM10 were principally localized in cytoplasm of tumor cells with varying staining intensity. A. High expression of ADAM10 (200x). B. High expression of ADAM10 (400x). C. Low expression of ADAM10 (200x). D. Low expression of ADAM10 (400x). E. Negative ADAM10 expression (200x). F. Partial enlargement of ADAM10 staining with the magnifying power of 400x.

Journal: Oncotarget

Article Title: Gemcitabine inhibits immune escape of pancreatic cancer by down regulating the soluble ULBP2 protein

doi: 10.18632/oncotarget.11780

Figure Lengend Snippet: The ADAM10 were principally localized in cytoplasm of tumor cells with varying staining intensity. A. High expression of ADAM10 (200x). B. High expression of ADAM10 (400x). C. Low expression of ADAM10 (200x). D. Low expression of ADAM10 (400x). E. Negative ADAM10 expression (200x). F. Partial enlargement of ADAM10 staining with the magnifying power of 400x.

Techniques: Staining, Expressing

Correlation between ULBP2 and ADAM10 expression and clinicopathological characteristics

Journal: Oncotarget

Article Title: Gemcitabine inhibits immune escape of pancreatic cancer by down regulating the soluble ULBP2 protein

doi: 10.18632/oncotarget.11780

Figure Lengend Snippet: Correlation between ULBP2 and ADAM10 expression and clinicopathological characteristics

Techniques: Expressing, Staining

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