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: Protein identity was confirmed on LS180-biotinylated material by 2DE-WB using an anti-ADAM10 Ab (AB936, R&D Systems).

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

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 1. Specificity of a-ADAM10 monoclonal antibodies. (A) Alignment of mouse, human and bovine ADAM10 cysteine-rich domain sequences (AA 551– 646). In the human and bovine sequences, only residues not homologous to mouse are shown. (B) Comparison of binding of mouse hybridoma (fusion) and isolated cell clone supernatants to serially diluted, immobilised bovADAM10 ECD by ELISA. Binding of non-immunised mouse serum (control) is shown for comparison. (C) Binding of endogenous huADAM10 by a-ADAM10 hybridoma clones, or the R&D ADAM10 mAb 1427, was compared by immunoprecipitation from equivalent HEK293 cell lysates and western blotting with an a-ADAM10 pAb; u, unprocessed; p, processed ADAM10. (D) The specificity of 8C7 for ADAM10 was tested by immunoprecipitation from lysates of ADAM10 knockout (2/2) and Wt (+/+) mouse embryonic fibroblasts (MEFs), and a-ADAM10 pAb western blot.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Bioprocessing, Comparison, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Control, Clone Assay, Immunoprecipitation, Western Blot, Knock-Out

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 2. Co-staining of cells with ADAM10 mAb 8C7 and ephrin-A5-Fc reveals colocalisation and co-internalisation with EphA3. (A) EphA3/ HEK293 cells were incubated on ice with Alexa647–8C7 mAb and fixed for imaging (0 min) or first allowed to warm to 37˚C for 60 min. (B) Cells were labelled with Alexa647–8C7 and with Alexa488–ephrin-A5-Fc and fixed immediately (0 min) or incubated at 37˚C with a-humanFc to cluster ephrin- A5-Fc for the indicated time periods before fixation. The insets are enlarged images of the regions within the dotted lines. Cells incubated for 60 min with Alexa488–ephrin-A5-Fc alone are shown as a control in the bottom panels. Scale bars: 25 mm.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Staining, Incubation, Imaging, Control

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 3. Site-directed mutagenesis of the ADAM10 substrate-binding pocket disrupts mAb binding. (A) Structure of the bovine ADAM10 D and C domains showing the location of key residues targeted by site-directed mutagenesis. (B) Comparison of aADAM10 mAb binding to Wt and substrate-binding pocket mutant huADAM10. Alanine substitutions at Glu 573, 578 and 579 (3EA) or at residues 617 and 618 (617AA) were made in huADAM10-GFP, and Wt and mutant constructs were transfected into ADAM102/2 MEFs (control: untransfected). Binding of a-ADAM10 mAbs was assessed by immunoprecipitation from equivalent cell lysates, and western blotting with a-ADAM10 pAb (non-relevant lanes removed; the altered molecular mass pattern reflects the GFP-tagged huADAM10). The graph shows binding of 8C7 and 3A8 relative to the R&D mAb, determined by densitometry (one-way ANOVA; **P,0.01 compared to R&D sample; n.s., not significant; n53).

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Mutagenesis, Binding Assay, Comparison, Construct, Transfection, Control, Immunoprecipitation, Western Blot

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 5. ADAM10 mAb 8C7 inhibits EphA3 phosphorylation in response to stimulation by cell-bound ephrin. (A) 293/EphA3 cells were pretreated with 0, 10 and 100 mg/ml of 8C7 mAb for 2 h and stimulated for the indicated times. a-EphA3 immunoprecipitates from the cell lysates were analysed by western blot with a-phosphotyrosine (pY) and a-EphA3 antibodies as indicated. A representative image from four experiments is shown. (B) EphA3 phosphorylation relative to EphA3 protein levels was calculated from replicate experiments as described in A, using densitometry analysis. Graph shows means 6 s.e.m., n54. (C) 8C7 does not inhibit EphA3 phosphorylation induced by soluble clustered ephrin-A5. EphA3/293 cells, pre-incubated with or without 8C7 (100 mg/ml) for 2 hours, were stimulated for 20 min with pre-clustered ephrin-A5-Fc, or left unstimulated, as indicated. EphA3 immunoprecipitates from cell lysates were analysed by western blotting as in A.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: Journal of cell science

Article Title: Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function.

doi: 10.1242/jcs.112631

Figure Lengend Snippet: Fig. 6. ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell repulsion. (A) EphB2/HEK293 cells labelled with Cell Tracker Green were pre-treated with vehicle (Cont), 8C7 (50, 200 or 400 mg/ml), or with GM6001 (GM, 50 mM), and plated onto coverslips pre-coated with fibronectin and stripes of alexa594-labelled ephrin-A5-Fc. As a comparison, cells expressing a signalling-deficient EphB2 mutant (DICD) were also used. After 18 hours the cells were imaged by fluorescence microscopy, from which examples are shown (8C7, 400 mg/ml). Scale bar: 250 mm. (B) The percentage of cells adhering to ephrin stripes was calculated from ,20 images for each treatment; the graph shows the averages 6 s.e.m. from three experiments. (C) 8C7 inhibits ephrin-A5-induced EphB2 phosphorylation. Effects of 8C7 treatment on activation of EphB2/HEK293 cells by ephrin-A5/HEK293 cells was assessed as in Fig. 5A, following stimulating for 40 minutes.

Article Snippet: Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al., 2002) were immunoprecipitated with antibodies against ADAM10 (R&D systems mAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A– Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated to MinileakTM beads).

Techniques: Comparison, Expressing, Mutagenesis, Fluorescence, Microscopy, Phospho-proteomics, Activation Assay

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: The mature membrane form of ADAM10 is expressed on HL cells. Panels (A), (B), (D) Lysates obtained from HL LN cell suspensions (A) or HL cell lines (B) or LN MSCs obtained by culturing LN cell suspensions from HL patients (D) were subjected to Western blot as described in Materials and Methods; membranes were probed with the anti-ADAM10 or anti-β actin mAb followed by the relevant HRP-conjugated secondary antibodies and developed with the HRP substrate. In each blot, the precursor form (p) and the mature form (m) of ADAM10 molecule is indicated. Panels (C) and (E) Surface expression of ADAM10 on KMH2, L540, L428 (Ca, Cb, Cc, dark gray histograms) or MSC773 or RS773 (Ea, Eb) was evaluated with the specific mAb directed against the mature form of ADAM10 followed by APC-conjugated GAM and FACS analysis; results are expressed as Log far red fluorescence intensity, a.u., vs. number of cells.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Membrane, Western Blot, Expressing, Fluorescence

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: ADAM10 silencing leads to decreased shedding of NKG2D-L. KMH2 and L428 cells were transfected with ADAM10 (siADAM10) or ADAM17 (siADAM17) siRNA or non-targeting siRNA (siNT) pool as negative control (KMH2: panels A, B; L428: panels C, D). Protein expression was analyzed by Western blot (panels Aa and Ca), and FACS analysis (Ab and Cb; in each histogram the percentage and MFI of positive cells is shown) with the specific anti-ADAM10 or anti-ADAM17 antibodies, 72 h after transfection. Soluble MIC-B (Ba, Da) or ULBP3 (Bb, Db) or sALCAM (Bc, Dc) were evaluated by ELISA in SN (collected upon further 24 h of culture 72 h after transfection). Results in (B) and (D) are expressed as pg/mL/10 5 cells and are the mean ± SD from three independent experiments. * p <0.001 vs siNT.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

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

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: In vitro enzymatic activity (IC 50 nM values) a of new compounds LT4 and MN8 and the reference compounds JG26 and GI254023X.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: In Vitro, Activity Assay

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: ADAM10 inhibitors reduce the shedding of NKG2D-L by HL cell lines and maintain the binding of NKG2D receptor. L428 cells were exposed to culture medium alone, DMSO or GI254023X (GIX), JG26, MN8 or LT4 (at 10 to 2.5 μM concentration) for 24 h (panel A), followed by 100 μM Na 3 VO 4 as pervanadate for 40 min at 37°C (panel B and C). Then, SN were harvested and sMIC-A (Aa, Ba), sMIC-B (Ab, Bb), sULBP2 (Ac, Bc), sULBP3 (Ad, Bd) or sALCAM (C) measured by specific ELISA. Results are expressed as ng/mL/10 6 cells and are representative of four independent experiments. * p <0.001 vs. DMSO. Panels (D) and (E) L428 cells exposed for 24 h to DMSO or 10 μM LT4 or 100 μM Na 3 VO 4 as pervanadate, in the absence or presence of 10 μM LT4 as indicated, were harvested and evaluated for the expression of ULBP2 (D) with the specific mAb followed by APC-conjugated GAM or for the binding of the chimeric receptor (FcNKG2D, panel E) followed by APC-conjugated anti-human Fc antiserum, by FACS analysis; results are expressed as Log far red fluorescence intensity (arbitrary units, a.u.) vs. number of cells. In each subpanel: percentage and mean fluorescence intensity (MFI, a.u.) of positive cells. One representative experiment out of four.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: Exposure to ADAM10 inhibitors increases the sensitivity of HL cell lines to NKG2D-dependent cell killing. Cytolytic activity of NK cells (n = 6, panel A and B) or γδ T cells (n = 6, panel C) was analyzed against L428 (Aa, Ab, Ca) or L540 (Ba, Bb, Cb) cell lines at E:T ratio of 10:1 in a 4-h 51 Cr-release assay. Some samples were set up after exposure of the target cell lines to either medium, or DMSO or LT4 or MN8 (Aa, Ba, Ca, Cb), GIX or JG26 (Ab, Bb) at 10 μM concentration for 24 h. In some samples, effector cells were exposed to saturating amounts (5 μg/mL) of the anti-NKG2D mAb at the onset of the cytotoxicity assay; an unrelated mAb, matched for the isotype, used as control, did not exert any effect (not shown). Results are expressed as % specific lysis calculated as described in Materials and Methods.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Release Assay, Concentration Assay, Cytotoxicity Assay, Control, Lysis

Journal: Oncoimmunology

Article Title: ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

doi: 10.1080/2162402X.2015.1123367

Figure Lengend Snippet: Improvement of HL cell lysis by exposure to ADAM10 inhibitor LT4 and anti-TGFβ. Panel (A) NKG2D expression before (upper histograms) or after treatment with TGFβ (10 ng/mL), (middle histograms) or with TGFβ and anti-TGFβ mAb (1 µg/mL), on NK cells (Aa) or γδ T cells (Ab). In each subpanel: percentage of positive cells and MFI (a.u.). Panel (B) Cytolytic activity of NK cells (Ba) orγδ T cells (Bb) was analyzed against L428 cell line at E:T ratio of 5:1 in a 4-h 51 Cr-release assay. Some samples were set up after exposure of the target cell lines to LT4 or MN8 at 10 μM concentration for 24 h. To some samples, we added effector cells exposed to TGFβ (10 ng/mL), with or without saturating amounts (1 μg/mL) of the anti-TGFβ mAb, as indicated. Results are expressed as % inhibition or stimulation of specific lysis calculated as described in Materials and Methods. * p <0.001 vs. TGFβ. ** p <0.001 vs. TGFβ + anti-TGFβ. # p <0.001 vs. TGFβ + anti-TGFβ on untreated L428 cells.

Article Snippet: Recombinant human ADAM10 was from R&D Systems (Minneapolis, MN).

Techniques: Lysis, Expressing, Activity Assay, Release Assay, Concentration Assay, Inhibition

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: The Journal of Biological Chemistry

Article Title: A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein *

doi: 10.1074/jbc.M113.502351

Figure Lengend Snippet: ADAM10 and ADAM17 both cleave MoPrP at Ala-119↓Val-120. A, MoPrP was reacted with ADAM10 and ADAM17 at both pH 7.4 and enzymatically optimal pH 9. ADAM10 and ADAM17 cleaved MoPrP at Ala-119↓Val-120, producing α3-N1 and α3-C1. ADAM10 also cleaved MoPrP at Gly-227↓Arg-228, producing the N3 fragment. B, even after 24 h, the mutant MoPrP V120D experienced greatly reduced α3-cleavage by both ADAM10 and ADAM17 at pH 7.4. ADAM10 cleavage at Gly-227↓Arg-228 remained at levels comparable to those of wild-type MoPrP. C, cleavage near the C terminus releasing the N3 fragment was ablated in MoPrP R228D, whereas cleavage at Ala-119↓Val-120 was unaffected.

Article Snippet: ADAM10 and ADAM17 Cleavage of MoPrP Recombinant human ADAM10 and ADAM17 were purchased from R&D Systems.

Techniques: Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein *

doi: 10.1074/jbc.M113.502351

Figure Lengend Snippet: PrP ΔCR does not produce α1-, α2-, or α3-cleavage fragments. PrP ΔCR was reacted with ADAM8 (left), ADAM10 (middle), or ADAM17 (right). The relative abundance of N-terminal cleavage fragments is shown. ADAM8 still cleaved ΔCR within the octarepeat domain (N2), but no α1- or α2-cleavage was observed. ADAM10 cleaved ΔCR at Gly-227↓Arg-228 (wild-type amino acid indexing), producing the N3 fragment, but cleavage at Ala-119↓Val-120 was abolished. Instead, a small amount of peptide fragment corresponding to cleavage at Tyr-127↓Met-128 was observed. ADAM17 also did not cleave ΔCR at Ala-119↓Val-120 but instead at Gly-130↓Ser-131.

Article Snippet: ADAM10 and ADAM17 Cleavage of MoPrP Recombinant human ADAM10 and ADAM17 were purchased from R&D Systems.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein *

doi: 10.1074/jbc.M113.502351

Figure Lengend Snippet: Schematic of PrP cleavage. The octarepeat domain (gold), the folded C-terminal domain (green), and C-terminal GPI anchor are shown. A, ADAM8 cleaves PrP both in the octarepeat domain (β-cleavage) and at Lys-109↓His-110 (α1-cleavage). The arrow thickness correlates to relative cleavage activity. B, upon addition of 2 eq of Cu2+, β-cleavage and α1-cleavage are diminished, whereas α2-cleavage is enhanced. C, the addition of Zn2+ results in diminished β-cleavage while maintaining similar levels of α1-cleavage relative to the apo-state. D, ADAM8, ADAM10, and ADAM17 all cleave MoPrP at distinct locations. The only overlap is at the α3-location, where both ADAM10 and ADAM17 cleave. E, the MoPrP mutant H110Y blocks ADAM8 cleavage at Lys-109↓Tyr-110 (α1-cleavage). F, the MoPrP mutant V120D blocks ADAM10 and ADAM17 cleavage at Ala-119↓Asp-120 (α3-cleavage); however, the ability of ADAM10 to cleave MoPrP V120D at Gly-227↓Arg-228 remains unaffected. G, MoPrP R228D blocks ADAM10 cleavage at Gly-227↓Asp-228 (resulting in the N3 fragment); however, ADAM10 still cleaves MoPrP R228D at Ala-119↓Val-120.

Article Snippet: ADAM10 and ADAM17 Cleavage of MoPrP Recombinant human ADAM10 and ADAM17 were purchased from R&D Systems.

Techniques: Activity Assay, Mutagenesis

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: Neonatal monocytes display elevated pro-AREG surface expression and increased release of AREG in response to E. coli infection. PBMO and CBMO were incubated with E. coli for 1 h, extracellular bacteria were removed, and cells were cultivated for 24 h in total. (a) Immunocytological staining shows AREG protein expression (green) in monocytes. Texas Red-X phalloidin was applied to visualize F-actin in the cytoskeleton (red). (b) Pro-AREG surface expression in uninfected and E. coli -infected monocytes was quantified by using flow cytometry ( n = 5). Representative dot plots show gating strategy and cutoff value for AREG expression. (c) sAREG levels in the supernatant of monocytes were quantified by using ELISA ( n = 5). Data are shown as means + SD. Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparisons test (ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Expressing, Infection, Incubation, Bacteria, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: AREG mediates increased shedding of memFasL through engagement with EGFR. PBMO and CBMO were infected as described in . AREG stimulation and EGFR inhibitor treatment was started 1 h prior to infection and was maintained during cultivation. (a) memFasL surface expression in uninfected and E. coli -infected monocytes was analyzed in response to AREG stimulation and neutralization of EGFR by using flow cytometry ( n = 5). AREG stimulation significantly decreased FasL levels in infected PBMO but not CBMO. Neutralization of EGFR resulted in a complete rescue of infection-induced memFasL presentation. Representative dot plots show gating strategy and cutoff value. (b) sFasL levels in the supernatant of uninfected and E. coli -infected monocytes were quantified in response to AREG stimulation and neutralization of EGFR by using ELISA ( n = 3). AREG significantly increased sFasL levels in infected PBMO but not CBMO, while neutralization of EGFR abolished the effect. Data are shown as means + SD. Statistical significance was analyzed using two-way ANOVA with Bonferroni's multiple comparisons test ( ∗∗ p < 0.01, ∗∗∗ p < 0.005, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Infection, Expressing, Neutralization, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and Matrix Metalloproteinases

doi: 10.1155/2018/4310419

Figure Lengend Snippet: EGFR-dependent gelatinase activation mediates increased shedding of memFasL. PBMO and CBMO were infected as described in . Stimulation with AREG and gelatinase inhibition by CHX treatment was started 1 h prior to infection and was maintained during cultivation. (a) memFasL surface expression in uninfected and E. coli -infected monocytes was analyzed in response to AREG stimulation and gelatinase inhibition by using flow cytometry ( n = 4). Gelatinase inhibition prevented the decrease in memFasL presentation on monocytes triggered by AREG. (b) sFasL levels in the supernatant of uninfected and E. coli -infected monocytes were quantified in response to AREG stimulation and CHX treatment by using ELISA ( n = 3). Gelatinase inhibition was found to prevent the increase in sFasL levels triggered by AREG. Data are shown as means + SD. Statistical significance was analyzed using two-way ANOVA with Bonferroni's multiple comparisons test ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.001).

Article Snippet: For detection of FasL, Human FasL PicoKine ELISA Kit (Boster Biological Technology, Pleasanton, USA) was used according to the manufacturer's instructions.

Techniques: Activation Assay, Infection, Inhibition, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

doi: 10.1242/jcs.060830

Figure Lengend Snippet: Detection of a faster-migrating BTC-ICD fragment in IMPE-BTC-WT cells upon constitutive and Ca2+-ionophore-induced BTC shedding. (A) Cell lysates were prepared from IMPE-BTC-WT and IMPE-Vector-alone (control) cells grown under serum-free conditions. (B) A431 cells were treated with or without the Ca2+ ionophore A23187 for 1 hour in the presence or absence of GI254023X (GI) prior to preparation of cell lysates. Left panel: cell lysates were directly analyzed by western blotting using an anti-human BTC cytoplasmic-domain antibody. Right panel: ionophore-induced A431 cell lysates were immunoprecipitated with control IgG or anti-BTC cytoplasmic-domain antibody prior to western blotting using the same antibody. Asterisks indicate IgG heavy (top) and light (bottom) chains. (C) IMPE-BTC-WT cells were treated with or without the Ca2+ ionophore A23187 for 1 hour in the presence or absence of GI254023X (GI) prior to preparation of cell lysates (upper panel: short exposure shows ionophore-induced ectodomain cleavage of BTC-FL and concomitant BTC-CTF production; lower panel: long exposure shows ionophore-induced generation of BTC-ICD). (D) IMPE-BTC-WT cells were treated overnight with or without metalloprotease inhibitor GI254023X or γ-secretase inhibitor PIX. CM was collected prior to preparation of cell lysates. Graph shows BTC shedding into CM, which was measured by BTC ELISA. Blot: all cellular BTC isoforms from IMPE cells were precipitated from cell lysates with anti-HA agarose and then analyzed in western blotting with anti-HA antibody.

Article Snippet: Antibodies and reagents The following antibodies and reagents were used: biotinylated goat anti-human BTC ectodomain antibody, mouse anti-human BTC ectodomain antibody and rat anti-mouse ADAM10 antibody (R&D Systems); rabbit anti-HA epitope tag antibody (Zymed Laboratories, Bethyl Laboratories); mouse anti-HA agarose beads (Sigma); mouse anti-GFP (Clontech); horseradish-peroxidase-conjugated donkey anti-rabbit IgG F(ab)2 fragment (Amersham Biosciences); mouse anti-actin anti-histone-2 (Millipore); and mouse anti-PolII antibody (Santa Cruz Biotechnology).

Techniques: Plasmid Preparation, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

doi: 10.1242/jcs.060830

Figure Lengend Snippet: ADAM10 is required for proBTC ectodomain cleavage and production of BTC-CTF and BTC-ICD. (A) WT MEFs or ADAM10−/− MEFs expressing proBTC were grown in serum-free DMEM for 24 hours in the presence or absence of GI254023X. BTC shedding into CM was measured by BTC ELISA. (B) WT MEFs and ADAM10−/− MEFs expressing proBTC were grown in serum-free DMEM for 24 hours in the presence or absence of GI254023X or PIX. Cell lysates were precipitated with anti-HA agarose and analyzed by western blot with anti-HA antibody. (C) WT MEFs or ADAM10−/− MEFs expressing proBTC were pre-incubated for 30 minutes in the presence or absence of GI254023X or PIX before treatment with or without A23187 for 1 hour. BTC shedding into CM was measured by BTC ELISA. (D) WT MEFs expressing proBTC were treated under the same conditions as in C. Cell lysates were precipitated with anti-HA agarose and analyzed by western blot with anti-HA antibody (upper panel: short exposure; lower panel: long exposure shows only BTC-CTF and BTC-ICD).

Article Snippet: Antibodies and reagents The following antibodies and reagents were used: biotinylated goat anti-human BTC ectodomain antibody, mouse anti-human BTC ectodomain antibody and rat anti-mouse ADAM10 antibody (R&D Systems); rabbit anti-HA epitope tag antibody (Zymed Laboratories, Bethyl Laboratories); mouse anti-HA agarose beads (Sigma); mouse anti-GFP (Clontech); horseradish-peroxidase-conjugated donkey anti-rabbit IgG F(ab)2 fragment (Amersham Biosciences); mouse anti-actin anti-histone-2 (Millipore); and mouse anti-PolII antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation