Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A , comparison of TspanC8 co-immunoprecipitations with ADAM10 truncation constructs. Quantitation of the co-immunoprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from were compared. Values were normalized using Tspan14 data from . All data were relative to the co-immunoprecipitation of ADAM10DCS with Tspan14, which was arbitrarily set to 100. Data were log transformed and statistical analysis was performed using a one-way ANOVA with a Dunnett's multiple comparison test comparing ADAM10CS (#, p < 0.01) or ADAM10S (*, p < 0.01) to the ADAM10DCS for each tetraspanin. Error bars represent the standard error of the mean from three experiments. B , schematic of the potential differential modes of interaction of the TspanC8s with ADAM10. Bold regions of ADAM10 represent those required for a strong interaction with the corresponding TspanC8. Note that Tspan15 has 3 N -linked glycosylation sites and Tspan17 has 2, whereas Tspan5, 10, 14, and 33 have 3, 0, 1, and 2, respectively; for the latter, Tspan14 is depicted as an example.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Comparison, Construct, Quantitation Assay, Immunoprecipitation, Transformation Assay, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The combined cysteine-rich ( C ) and stalk ( S ) region of ADAM10 without the disintegrin ( D ) is sufficient to interact with Tspan14. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of Myc-tagged human ADAM10, or pDisplay constructs containing ADAM10DCS or ADAM10CS, which also possessed Myc tags. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). B , data in panel A ( upper panel ) were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett's multiple comparison test against the mock. Tspan14 bound significantly to ADAM10DCS ( p < 0.0001) and ADAM10CS ( p < 0.0001). A diagrammatic representation of the ADAM10 constructs is shown below the graph. C , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged human CD9 or Tspan14, with co-transfection of pDisplay ADAM10CS or ADAM10S. Cells were treated as in panel A. D , data in panel C were quantitated from three experiments. Data were log transformed and compared statistically with a one-way ANOVA with a Dunnett's multiple comparison test against the mock. Tspan14 bound significantly to ADAM10CS ( p < 0.0001) and ADAM10S ( p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Cotransfection, Construct, Lysis, Immunoprecipitation, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The TspanC8s bind differentially to the disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse TspanC8s or CD9, and co-transfected with the pDisplay vector containing HA-tagged human ADAM10DCS. Cell lysates were produced in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). B , data from panel A ( upper panel ) were quantitated and presented as the amount of immunoprecipitated ADAM10DCS relative to the Tspan14 immunoprecipitation, which was arbitrarily set to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the CD9 control. All TspanC8s bound significantly to ADAM10DCS ( p < 0.001). Error bars represent the standard error of the mean from three experiments. C and D , these experiments were carried out as described for panels A and B except using HA-tagged human ADAM10CS. All TspanC8s bound significantly to ADAM10DCS ( p < 0.0001). E and F , these experiments were carried out as for panels A and B except using HA-tagged human ADAM10S (****, p < 0.0001; **, p < 0.01; *, p < 0.05).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Plasmid Preparation, Produced, Lysis, Immunoprecipitation, Transformation Assay, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Endogenous ADAM10 and Tspan14 interact in platelets and primary endothelial cells. A , HEK-293T cells were mock transfected (−) or transfected with a FLAG-tagged human Tspan14 expression construct (+). The cells were lysed in 1% Triton X-100 lysis buffer and subjected to anti-Tspan14 ( top panel ) and anti-FLAG ( lower panel ) Western blotting. The Tspan14 antibody was raised in goat against a C-terminal cytoplasmic peptide, in collaboration with Everest Biotech. B , washed human platelets; C , washed mouse platelets and D , human umbilical vein endothelial cells were lysed in 1% digitonin lysis buffer, and proteins were immunoprecipitated with an antibody against ADAM10 or an isotype-matched control. Precipitates were then run on non-reducing gels, Western blotted, and probed with Tspan14 ( top panels ), ADAM10 ( middle panels ), and CD9 ( lower panels ) antibodies. Arrows indicate the positions of the predominant mature form of ADAM10 (A10) and the signal from the immunoprecipitating antibodies (IgG).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Expressing, Construct, Lysis, Western Blot, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A , schematic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable (var) region of CD9 ( black ) and Tspan14 ( gray ) were interchanged; the N -linked glycosylation site of Tspan14 is indicated by a filled oval. B , HEK-293T cells were mock transfected (−) or transfected with expression constructs containing the FLAG-tagged human tetraspanin chimeras with Myc-tagged human ADAM10 (+). Cell lysates were produced using 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-Myc tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-Myc tag antibody ( middle panel ). Data are representative of three independent experiments. C , quantitation of immunoprecipitated ADAM10. Data in panel B ( upper panel ) were quantitated using the Odyssey Infrared Imaging System (LI-COR), and the amount of ADAM10 immunoprecipitated was shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock (****, p < 0.0001). Error bars represent standard error of the mean from three experiments. D , data in panel B ( middle panel ) were quantitated, the percentage of mature ADAM10 calculated, and the data log transformed and statistically analyzed as described for panel C (***, p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Glycoproteomics, Transfection, Expressing, Construct, Produced, Lysis, Immunoprecipitation, Quantitation Assay, Imaging, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The large extracellular loop (LEL) of Tspan14 is critical for its ability to increase ADAM10 cell surface accumulation. A , HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see A ) and GFP to identify transfected cells. Cells were stained with an APC-conjugated ADAM10 antibody and analyzed by flow cytometry. Dot plots are representative of three independent experiments. The bottom left panel shows isotope control staining. B , average geometric mean fluorescent intensities for ADAM10 staining, gated on live and GFP-positive cells, were compared statistically using a one-way ANOVA with a Dunnett's multiple comparison test, compared with the CD9 control (***, p < 0.001; **, p < 0.01). Error bars represent standard error of the mean from three experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Flow Cytometry, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: All Tspan14-CD9 chimeras partially co-localize with ADAM10 and so have access to the metalloprotease. HeLa cells were transfected with the indicated Tspan14-CD9 chimeras (see A ) and HA-tagged mouse ADAM10. Cells were fixed and stained with an anti-HA antibody ( green ) and an anti-FLAG antibody ( red ). Confocal microscopy images are representative of three independent experiments and at least 15 fields of view.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The region of ADAM10 comprising the disintegrin domain ( D ), the cysteine-rich ( C ), and stalk ( S ) regions mediates the interaction with Tspan14. A , schematic of ADAM10 and ADAM17 chimeras. The extracellular disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10 ( black ) and ADAM17 ( gray ) were interchanged together ( DCS ) or individually. B , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse Tspan14 (+) in addition to either HA-tagged mouse ADAM10, ADAM17, ADAM17 10DCS, or ADAM10 17DCS. Cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with an anti-FLAG antibody. Immunoprecipitated proteins were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-HA tag antibody ( middle panel ). The blots are representative of three independent experiments. C , HEK-293T cells were co-transfected with (+) or without (−) FLAG-tagged mouse Tspan14 and either HA-mouse ADAM10, ADAM17, ADAM17 10DCS, ADAM17 10D, ADAM17 10C, or ADAM17 10S. Cells were treated as in B. D , data from panels B and C were quantitated and presented as the relative amount of each ADAM10/17 construct immunoprecipitated with Tspan14, having arbitrarily set wild-type ADAM10 to 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test, compared with the ADAM17 control (*, p < 0.05). Error bars represent standard errors of the mean from 3–6 experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Lysis, Immunoprecipitation, Construct, Transformation Assay, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: All TspanC8s interact with the region of ADAM10 comprising the disintegrin ( D ), cysteine-rich domain ( C ), and stalk ( S ). A , HEK-293T cells were transfected with expression constructs for the HA-tagged mouse ADAM17 10DCS chimera and FLAG-tagged mouse TspanC8s, CD9 or negative control (−). Lysates were extracted in 1% digitonin lysis buffer and proteins immunoprecipitated with an anti-FLAG antibody. Immunoprecipitates were blotted with anti-HA tag antibody ( top panel ) or anti-FLAG antibody ( lower panel ). Whole cell lysates were probed with the anti-HA tag antibody ( middle panel ). B , data in panel A ( upper panel ) were quantitated, and the amount of ADAM17 10DCS immunoprecipitated was normalized for the amount in the whole cell lysate. Data are shown relative to immunoprecipitated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock. All TspanC8s bound significantly to ADAM17 10DCS ( p < 0.0001). Error bars represent standard error of the mean from three experiments. C , ADAM17 10DCS whole cell lysate data in panel A were quantitated, and the amount of ADAM17 10DCS expressed was normalized to the expression in the first lane, which was arbitrarily set at 100. Error bars represent standard error of the mean from three experiments.

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Expressing, Construct, Negative Control, Lysis, Immunoprecipitation, Transformation Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: The disintegrin ( D ), cysteine-rich ( C ), and stalk ( S ) regions of ADAM10 are essential for Tspan14-mediated exit from the ER. A , HeLa cells were transfected with combinations of FLAG-tagged Tspan14 and HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were fixed and stained with an anti-HA antibody ( green ), an anti-FLAG antibody ( red ) and WGA to visualize the plasma membrane and internal cellular structures by confocal microscopy. B , HeLa cells were transfected and stained as in panel A except an anti-calnexin antibody was used instead of WGA to define the limits of the ER (images not shown). The HA signal was quantitated across the whole cell and within the mask of the calnexin staining, and presented as a percentage of HA-ADAM10 or HA-ADAM10 17DCS signal localized in the ER. Data are representative of three independent experiments and at least 15 fields of view. A two-way ANOVA statistical analysis was performed with a Bonferroni's multiple comparisons test ( ns , non-significant, ****, p < 0.0001). C , HEK-293T cells were mock transfected (−), or transfected with HA-tagged mouse ADAM10 wild-type or ADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HA antibody. Immunoprecipitates were stained with neutravidin ( top panel ) or an anti-HA antibody ( bottom panel ). Whole cell lysates were stained with an anti-HA antibody ( middle panel ).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Clinical Proteomics, Membrane, Confocal Microscopy, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

doi: 10.1074/jbc.M115.703058

Figure Lengend Snippet: Differential effects of TspanC8s on ADAM10 substrate cleavage: Tspan15 promotes cleavage of N-cadherin and Tspan14 reduces cleavage of GPVI. A , HEK-293T cells were mock transfected (−) or transfected with FLAG-tagged mouse TspanC8s. The cells were lysed in 1% Triton X-100 lysis buffer and subjected to Western blotting with an antibody to the C-terminal cytoplasmic tail of N-cadherin ( upper panel ) or with an antibody to the FLAG epitope ( lower panel ). B , data from A ( upper panel ) were quantitated and the lower, cleaved band given as a percentage of the total ( upper and lower band combined). Data were normalized by log transformation and statistically analyzed using a one-way ANOVA with a Dunnett's multiple comparison test compared with the mock control. Error bars represent the standard error of the mean from three experiments (*, p < 0.05). C , HEK-293T cells were co-transfected with GPVI and FcRγ and one of each of the FLAG-tagged mouse TspanC8s or without a tetraspanin (−) or with the addition of the ADAM10 inhibitor GI254023X at 10 μ m . Cells were treated as in panel A , except lysates were subjected to an anti-GFP antibody ( upper panel ) instead of an anti-N-cadherin antibody. D , data from panel C ( upper panel ) were quantitated as described in panel A (***, p < 0.001).

Article Snippet: For Western blotting immunoprecipitation and immunofluorescence microscopy, primary antibodies were mouse anti-FLAG (M2) and rabbit anti-FLAG (Sigma), rabbit anti-HA (Cell Signaling Technologies (CST)), mouse anti-Myc (9B11) and rabbit anti-Myc (CST), mouse anti-human ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) , mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Lysis, Western Blot, FLAG-tag, Transformation Assay, Comparison, Control

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. Serological reactivity of Crc patient pool of sera and control pool of sera against the biotinylated protein spot (Av-HRP) that, from the preparative 2DE gel (Coomassie blue), yielded the MS identification of ADAM10; the identity was confirmed by reactivity of an anti-ADAM10 Ab with the same spot. B. Surface expression of ADAM10 in the LS180 Crc cell line. Anti-ADAM10 immunofluorescence reactivity is present on both permeabilized and non-permeabilized cells. Anti-HLA-class I and anti- ß-actin reactivities were used as controls for surface and intracellular expressed proteins, respectively. C. Reactivity of Crc patients and control subjects (Cn) sera against purified ADAM10; anti-ADAM10 Ab reactivity was used for signal normalization. D. - E. Quantitative analysis of serological reactivity reported as normalized OD (mean +/− SEM of 3 experiments in duplicate). D. Testing cohorts Crc1, n = 57; Cn1, n = 39; Crc1-stage I n = 8, stage II n = 17, stage III n = 26, stage IV n = 6. E. Validation cohorts Crc2, n = 49; Cn2, n = 52; Crc2-stage I n = 13, stage II n = 13, stage III n = 13; stage IV n = 10. Statistical analysis was performed by either student-t (S-t) test or Mann-Whitney (M-W) test and non parametric analysis of variance by Kruskal-Wallis (K-W). (*** = p < 0.0001; ** = p < 0.01).

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Control, Expressing, Immunofluorescence, Purification, Biomarker Discovery, MANN-WHITNEY

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. - D. Learning of the cut-off values for sera categorization (anti-ADAM10 Ab-positive or -negative) performed by ROC analysis of anti-ADAM10 optical density in Crc and Cn. In order to avoid over-fitting, the learning of the cut-off value (that was be applied to the group of patients in a specific stage of the disease) was done using all Crc patients excluding the group of patients at the stage that have been subsequently tested for the follow-up (Crc out Stage … = Crc without patients at specific stage). E. - H. Kaplan-Meier survival curve and Log-rank test analysis in the Crc patients at different stages. The analysis compares the patients showing immunoreactivity against ADAM10 (Ab-pos, solid line) vs. the patients with no reactivity (Ab-neg, dotted line). The considered outcome was recurrence-free survival (RFS) (patient alive, no relapse, no appearance of new metastasis), and the marked “events” were tumor relapse, metastasis and patient death. The dotted red line in panel G indicates the median RFS time significantly changing (from 23 to 55 months) in patients at stage III that showed immunoreactivity against ADAM10.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques:

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Cox regression analysis of outcome predicting factor ^°

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques:

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Immunohistochemistry analysis of ADAM10 expression in normal intestinal epithelia tissue a. and tumoral tissue b. - f. . Arrows indicated in panel a. the few ADAM10 positive monocytes resident in the cryptae, and in panels a. - f. the stronger reactivity of tumor cells invading the stroma.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Immunohistochemistry, Expressing

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: A. Anti-ADAM10 WB analysis in Crc specimens from representative patients with (Ab-pos, C27, C38, C9, C21, C36) or without (Ab-neg, C32, C52) serological reactivity against ADAM10. Anti-β-actin reactivity was used for protein-loading control and OD normalization. B. Quantitative analysis of WB reactivity detected on Ab-pos ( n = 15) and Ab-neg ( n = 12) patient specimens (mean +/− SEM 3 experiments; student's t -test; ** p < 0.01; *** p < 0.001). The ratio between pro-protein and mature ADAM10 signals is reported.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Control

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: ADAM10 pro-domain: reactivity of the rabbit polyclonal anti-ADAM10 pro-domain-specific Ab (ab39178, Abcam), which recognized the immature non-functional isoform of ADAM10, showed patched reactivity. ADAM10 ectodomain: reactivity of the goat polyclonal anti-ADAM10 ectodomain-specific Ab (R&D-Systems, AB936), which recognized both the immature and mature (cleaved and functional) isoforms of ADAM10, showed patched and diffuse signals. Anti-ADAM10 auto-Ab positive serum: reactivity of the human IgG fraction purified from a representative serum (C2) of Crc patients considered positive for the presence of auto-Ab anti ADAM10, showed patched reactivity. Anti-ADAM10 auto-Ab negative serum: no reactivity was observed for the human IgG fraction purified from serum (C25) of a representative Crc patient considered negative for the presence of auto-Ab anti ADAM10. Cell nuclei were stained with Hoechst-33342. The reactivity of the secondary antibodies (goat anti-rabbit IgG Alexa-488; donkey anti-goat IgG Alexa-488 and goat anti-human IgG FITC) are shown as negative controls in . Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Functional Assay, Purification, Staining, Immunofluorescence, Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Double staining was performed with rabbit anti-ADAM10 pro-domain (ab39178, Abcam), and either IgGs purified from Crc patient serum or mouse anti-HLA class I (Santa-Cruz Biotechnology). Secondary Abs were Alexa-546-conjugated goat-anti-rabbit IgG and Alexa-488-conjugated rabbit-anti-mouse IgG or FITC-conjugated goat-anti-human IgG. Cell nuclei were stained with Hoechst-33342, and differential interference contrast (DIC) images were also acquired. Staining was assessed by immunofluorescence confocal microscopy (Leica TCS SP5 Laser Scanning Confocal) and images acquired with LAS-AF (Leica) software; magnification 63X. Images from single channel and merged images are shown. Images were linearly adjusted for brightness and contrast using ImageJ 1.47v software.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Double Staining, Purification, Staining, Immunofluorescence, Confocal Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: Anti-ADAM10 pro-domain: reactivity of the rabbit anti-ADAM10 pro-domain Ab (ab39178, Abcam) after competition with control rabbit IgG. Anti-ADAM10 positive serum (C2): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered positive for the presence of auto-Abs anti ADAM10 after competition either with control rabbit IgGs or the anti-ADAM10 pro-domain Ab. Anti-ADAM10 negative serum (C32): reactivity of the IgG fraction purified from a representative serum of a Crc patient considered negative for the presence of anti ADAM10 auto-Ab after competition either with control rabbit IgGs or anti-ADAM10 pro-domain Ab. Cell nuclei were stained with Hoechst-33342; secondary Abs were goat anti-rabbit IgG Alexa-488 and goat anti-human IgG FITC. Images were acquired by immunofluorescence microscopy (Zeiss Upright Axo Imager 2 equipped with AxoVision Rel.4.8.2 software); magnification 63X. Images were linearly adjusted for brightness and contrast using Adobe-Photoshop CS4 v.11 software.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Control, Purification, Staining, Immunofluorescence, Microscopy, Software

Journal: Oncotarget

Article Title: Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

doi: 10.18632/oncotarget.11181

Figure Lengend Snippet: The reactivity of the IgG fraction purified from the sera of 6 Crc patients positive for the presence of auto-Abs anti-ADAM10 (C2, C27, C20, C9, C14, C19) and 6 Crc patients considered negative (C32, C50, C25, C11, C13, C45) was tested on human-recombinant ADAM10 (mature form lacking the pro-domain), mouse-recombinant ADAM10 (immature form including the pro-domain), ADAM10 pro-domain synthetic peptide (32 aa within the pro-domain sequence), human-recombinant ADAM17 (immature form including the pro-domain) and purified human ceruloplasmin (Cp) proteins spotted (200 ng/spot) onto nitrocellulose membrane. The reactivity of the anti-ADAM10 ectodomain Ab that recognizes both mature and immature ADAM10, anti-ADAM10 pro-domain Ab, anti-ADAM17 and anti-ceruloplasmin Abs were used as controls.

Article Snippet: Each serum was tested twice and, after background subtraction, OD values were normalized to the reactivity of the anti-ADAM10 ectodomain Ab (AB936, R&D Systems) obtained in the same film exposure.

Techniques: Purification, Recombinant, Sequencing, Membrane

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: The ADAM10 multimodular structure and its different forms. ( A ) Furin/PC7 proteases cleave Pro-ADAM10, the inactive protein containing a pro-domain (85 kDa), to activate the enzyme during the transit through the Golgi compartment. The full-length active form (ADAM10 FL, 60–65 kDa) is then directed to the cell membrane where it works as sheddase. ADAM10 itself can be subject to ectodomain shedding, a process that leads to the formation of the soluble (sADAM10, 50–55 kDa) and membrane-anchored C-terminal domain (ADAM10 CTF, 10 kDa). Antibody binding sites used in this study to recognize ADAM10 protein. ( B ) Western blotting membranes using the ProSci 2051 antibodies (detection of the C-terminal region) in platelets and Abcam 39153 (detection of the N-terminal region) in plasma and CSF. MK: standard marker. ( C ) Western Blot analysis of 10 μg and 20 μg aliquots of plasma obtained from two control subjects (P1 and P2). Both ADAM10 antibodies recognize a band with an apparent molecular weight of 50 kDa.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Membrane, Binding Assay, Western Blot, Clinical Proteomics, Marker, Control, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Sociodemographic, clinical variables and neuropsychological evaluations of subjects in the groups.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Western blotting assays and ADAM10 protein quantification in CSF and plasma. ( A ) Western blotting and quantification of CSF and ( B ) plasma samples from controls (cognitively healthy), aMCI and mild AD participants probed against anti-N-terminal ADAM10 antibodies, which recognized a 50 kDa form of the protein. Serum albumin (66 kDa) from all participants was used as load control for the quantifications. The last lanes of plasma membranes were loaded with young control samples (red squares). * p = 0 .02; ** p = 0.01 (control vs mild AD). One-Way ANOVA and Mann-Whitney test. Graph Pad Prism 8.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Western Blot, Clinical Proteomics, Control, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: ADAM10 Plasma and CSF Levels Are Increased in Mild Alzheimer’s Disease

doi: 10.3390/ijms22052416

Figure Lengend Snippet: Analysis of ADAM10 activity in SH-5YSY neuroblastoma cells, platelets or CSF samples. ( A ) Western blotting analyses of subcellular fractions of SH-SY5Y human neuroblastoma cells; samples were probed for ADAM10 (immature form 85 kDA, mature form 65 kDa), GAPDH (37kDa) and Histone H3 (17kDa), Calnexin (90 kDa) and APP (106-110 kDa). ( B ) Graph reporting ADAM10 activity monitored for 60 min of reaction in the same fractions described in ( A ). The activity of ADAM10 was evaluated using a fluorogenic peptide as described in methods. ( C ) ADAM10 activity in CSF (rightmost graph) and in plasma (leftmost graph) of control cognitively healthy (control), amnestic mild cognitive impairment (aMCI) and mild Alzheimer’s disease (Mild AD) groups. The ADAM10 activity was also analyzed in SH-5YSY neuroblastoma cells, platelet samples and recombinant ADAM10. A specific inhibitor for ADAM10 GI254023X was incubated with platelets before the addition of substrate to demonstrate that the majority of the activity in samples was due to ADAM10 (* p < 0.0001). Two-Way ANOVA and Kruskal-Wallis test. Graph Pad Prism 8.

Article Snippet: After gel electrophoresis, the proteins were transferred to nitrocellulose membranes (Sigma-Aldrich, San Louis, MO, USA) using the mini trans-blot cell transfer system (BioRad, Hercules, CA, USA) for 1 h. The membranes were incubated with rabbit anti-ADAM10 primary antibody (cat. n. 2051, ProSci Poway, CA, USA and cat. n. 39153 Abcam, Cambridge, United Kingdom), followed by incubation with appropriate secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, cat. n. 97051, Santa Cruz Biotech Dallas, TX, USA).

Techniques: Activity Assay, Western Blot, Clinical Proteomics, Control, Recombinant, Incubation

Journal: Neural Regeneration Research

Article Title: Neuronal DJ-1 regulates microglial activation in Parkinson’s disease

doi: 10.4103/nrr.nrr-d-24-01047

Figure Lengend Snippet: Figure 3 ｜DJ-1 regulates ADAM10 maturation and trafficking to facilitate CX3CL1 release. (A, B) Western blot analysis showing the ratios of mature to precursor ADAM10 and ADAM17 in SH-SY5Y cells, with and without Park7 KD and ADAM10 rescue. (C) Immunofluorescence of ADAM10 (red, Alexa Fluor 594) in SH-SY5Y cells, with and without Park7 KD and ADAM10 rescue. Scale bar: 40 μm. (D) ELISA of CX3CL1 secretion in SH-SY5Y cells, with ADAM10 KD and with ADAM10 rescue or with Park7 in neurons. (E) Immunofluorescence of TH-positive neurons (green, Alexa Fluor 488) and ADAM10 (red, Alexa Fluor 594) in the SN of PD model brains from WT, Park7 KO, and Park7 KO + rescue mice. ADAM10 expressed in neurons: ADAM10 merged TH created yellow. Scale bar: 50 μm. (F) Western blotting of ER and plasma membrane fractions of brain tissues of WT and Park7 KO mice (G) and primary neurons, with and without Park7 KD (G), showing mature ADAM10 levels at the plasma membrane. Data expressed as means ± SEM (n = 6 per group in vivo experiments, n = 3 per group in vitro); *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using one- way analysis of variance followed by Tukey’s post hoc test (B–E) or Student’s t-tests (F, G). ADAM10: A metalloproteinase domain-containing protein 10; ADAM17: metalloproteinase domain-containing protein 17; DJ-1: protein deglycase DJ-1; ER: endoplasmic reticulum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; KD: knockdown; KO: knock out; NC: negative control; ns: no significance; Park7: Parkinson’s disease protein 7; PD: Parkinson’s disease; TH: tyrosine hydroxylase; WT: wild type.

Article Snippet: For dopaminergic neuron labeling, sections were treated with chicken antityrosine hydroxylase (TH) polyclonal antibody (1:4000 dilution; Abcam, Cambridge, Cambridgeshire, UK; Cat# ab76442, RRID: AB_297840) and ADAM10 antibody (1:100; Proteintech, Wuhan, Hubei, China; Cat# 25900-1- AP, RRID: AB_288029) at 4°C for 24 hours, followed by incubation with Alexa Fluor 594-conjugated goat anti-chicken IgG (1:1000; Abcam; Cat# ab150172, RRID: AB_3662878) at 4°C overnight.

Techniques: Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Membrane, In Vivo, In Vitro, Knockdown, Knock-Out, Negative Control