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: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 1. Generation of human Tspan15-expressing MEFs as an immunogen and validation of resulting mouse anti-human Tspan15 mAbs. (A) ADAM10-knockout MEFs (–) and ADAM10- knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–),

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Construct, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 3. Tspan15 mAbs 1C12 and 4A4 partially inhibit ADAM10/Tspan15 activity. (Ai) Wild-type (WT), ADAM10-knockout (A10 KO) and Tspan15-knockout (T15 KO) HEK-293T cells were transfected with a VE-cadherin expression construct. Cells were treated with 10 μM DAPT to prevent post-ADAM10 proteolysis by γ-secretase, followed by 2 mM NEM for 30 minutes to activate ADAM10. Cells were lysed in 1% Triton X-100 lysis buffer and subjected to western blotting with an antibody against the cytoplasmic tail of VE-cadherin. No C-terminal fragment was detected in the absence of NEM (data not shown). (Aii) VE-cadherin cleavage data were quantitated to calculate the percentage cleaved. Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 compared to WT). Error bars represent standard error of the mean from three independent experiments. (B) Wild-type HEK-293T cells were transfected with VE-cadherin, treated with Tspan15 mAbs or MOPC-21 negative control mAb for 30 minutes, and stimulated with NEM as described for panel

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Activity Assay, Knock-Out, Transfection, Expressing, Construct, Lysis, Western Blot, Transformation Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 4. Tspan15 and ADAM10 co-localise on the cell surface. (Ai) A549 cells were fixed and stained with anti-ADAM10 mAb (red) and either anti-Tspan15 mAb 5D4 (green) or anti-CD9 mAb 1AA2 (green). ADAM10, Tspan15 and CD9 on the basal membrane were imaged using TIRF microscopy. Images shown are representative of 48 fields of view from four independent experiments (scale bar 10 µm). (Aii) The degree of co-localisation between ADAM10 and Tspan15 or CD9 was determined using Manders’ coefficients to measure the proportion of overlapping pixels contained within total ADAM10 signal in the red channel (M1) and total Tspan15 or CD9 signal in the green channel (M2). Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test to compare M1 and M2, within and between Tspan15 and CD9 (***p<0.001 for all pairwise comparisons). Error bars represent standard error of the mean.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Staining, Membrane, Microscopy, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 5. ADAM10 is the principal Tspan15-interacting protein in HEK-293T cells. Wildtype (WT) and Tspan15-knockout (KO) HEK-293T cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with Tspan15 mAb 1C12 cross-linked to protein G sepharose beads. Proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Proteomic profiles of WT and Tspan15 KO HEK-293T immunoprecipitates are presented in a volcano plot to identify differentially expressed proteins. The minus log10 transformed p-value of each protein was plotted against the log2 transformed protein label free quantification ratio between the Tspan15 co-immunoprecipitation of WT samples and the control co-immunoprecipitation of Tspan15 KO samples. Proteins with significant fold change (p<0.05) are depicted in red; blue dots represent proteins with no significant changes in expression. A permutation-based false discovery rate estimation was applied and visualised as hyperbolic curves in grey.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Knock-Out, Lysis, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 6. Tspan15 protein expression requires ADAM10. (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 7. The requirement of Tspan15 for ADAM10 surface expression is cell type dependent. (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in Figure 4A. (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in Figure 6A.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 8. ADAM10 and Tspan15 form dynamic bimolecular fluorescence complementation (BiFC) complexes. (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (***p<0.001).

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Fluorescence, Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 9. A synthetic ADAM10/Tspan15 fusion protein is a functional scissor. (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: (A) Human keratinocytes (HaCaT), or mouse keratinocytes (PDV) were left untreated or incubated with S. aureus α-toxin (50 nM or 250 nM) for 2h, washed, and analyzed by FACS for exposure of phosphatidylserine at the outer leaflet of the plasma membrane (staining with annexinV-AlexaFluo647, Invitrogen, A23204), and for membrane damage, using PI. Bars indicate percent positive cells (Annexin V, PI, both). Data are mean values from four independent experiments, error bars indicate ± SEM. (B) HAP1ADAM10KO cells were mock transfected (H 2 O), or transiently transfected with plasmids encoding for bovine or murine wild type ADAM10. The next day quadruplicate samples were treated, or not, with 500 ng/ml α-toxin (16.6 nM) for 2h, before measuring cellular ATP. Shown are mean values of three independent experiments ± SEM; ** indicates significance between expression-plasmid transfections and mock transfection (H 2 O), with an adjusted P value of 0.0079 in a one-way ANOVA and Dunnett’s multiple comparisons; no significance (n.s.) was found for the comparison of mADAM10 vs. mock transfection. (C) Western-blot for detection of ADAM10 (antibody A124695) in whole cell lysates from cells, transfected as in (B); arrows on the right side indicate processed ADAM10 (upper), and mature ADAM10, respectively; note strong bands in samples with bADAM10 only.

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: Incubation, Staining, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: (A) Position N° 1 through 8 indicated below ADAM10ΔPD in this schematic highlight the eight amino acid residues in the mouse sequence, which deviate from both bADAM10 and hADAM10. (B) The table specifies the amino acid exchanges at positions numbered 1-8 in (A). Green numbers indicate those positions, changed in one of two compound mutants, black numbers, and the red “7” (corresponding to E665Q), indicate positions changed in the second compound mutant. (C) Left: Schematic showing bADAM10 truncation constructs, compound mutations or single residue mutations tested for their ability to confer to HAP1ADAM10KO cells susceptibility to α-toxin, when co-transfected with bovine PD (indicated by “+” on the left side). Samples receiving PD or ΔPD alone (two uppermost constructs) served as controls. Scale at the top indicates amino acid residue numbers in bADAM10. Color code of domains is as in (A). Right: Bars indicate ATP levels in HAP1ADAM10KO cells, which were (co)-transfected as indicated in the figure and treated with 500 ng/ml α-toxin for 2h relative to samples receiving no toxin. Data are from n ≥ 3 independent experiments; mean values ± SEM. Mutations at position N°7 (corresponding to E665 in bADAM10) are highlighted in red; these constructs, when co-transfected with PD, failed to confer to HAP1ADAM10KO cells sensitivity to α-toxin; the difference to H 2 O was not significant in a one way ANOVA with Tukey’s multiple comparisons. Similarly, no significant differences were found for NLNN, NLNNQ, ΔPep, ΔPep-Dis, ΔCys and stalk . In contrast, the compound mutation with changes at positions N° 2.3.4.6 (highlighted in green) plus PD, or mutant N°8 plus PD were fully active, like wild type ΔPD plus PD. **** indicates P < 0.0001 for comparisons with H 2 O control. (D) Western-blot using antibodies directed against the HA-Tag, for verification of expression of ADAM10-constructs indicated in the figure; arrows indicate ΔPD(-variants) (upper arrow), and PD (lower).

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: Sequencing, Mutagenesis, Construct, Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: (A) Plot of FACS-analysis of HAP1ADAM10KO cells transfected with bADAM10E665Q or bADAM10Q734P and subsequently stained for surface-expression of ADAM10 as detailed in the Methods section. Plots on the left hand show forward scatter vs . side scatter, plots on the right hand fluorescence intensity (FITC-A, log scale) vs. number of events. The two uppermost dot plots are controls w/o antibody, or with antibody, but no transfection of ADAM10. The two lower plots on the right hand document similar surface expression of bADAM10E665Q and bADAM10Q734P. (B) Summary of data from three independent experiments as in (A). Mean values ± SEM. The difference between the two constructs was not significant in a two-tailed Student’s t-test.

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: Transfection, Staining, Expressing, Fluorescence, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: (A) Grey box below the schematic of ADAM10 domain organization displays the sequence of the stalk ( purple) region and sequences of wild type (wt) and mutant ADAM10-derived peptides below. “E” labeled in green in the wt peptide sequence corresponds to residue E665 of hADAM10 or bADAM10. In mutant peptide, glutamic acid (E) was changed to lysine (K). (B) hemolysis of RRBC, incubated for 30 min at 37°C with indicated doses of S. aureus α-toxin in the presence of solvent or peptides (10 μM). Data are background-substracted OD. Mean values ± SEM from n=6 independent experiments. At α-toxin concentrations of 125 nM, 62.5 nM and 31.25 nM, hemolysis was significantly lower in the presence of wild type peptide as compared to samples receiving mutant peptide; adjusted P -values: 0.011, 0,015 and 0.03, respectively (two-way ANOVA and Tukey’s multiple comparison test). (C) Hypothetical model of ADAM10-ectodomain (hADAM10) including the stalk region (magenta); green arrow points to the side chain of E665, highlighted in green. The model was created using the I-TASSER server (Yang, Yan et al. 2015), see Methods section. A high-resolution structure of a large portion of the ectodomain has been published (Seegar, Killingsworth et al. 2017), and the corresponding part of the model of the present figure essentially matches that structure; the structure of the stalk region is uncertain.

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: Sequencing, Mutagenesis, Derivative Assay, Labeling, Incubation

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: (A) In silico docking of α-toxin monomer (red, pre-stem-region in cyan) to the hADAM10 ectodomain (blue) including the stalk region (green). For details see Methods section. The yellow arrow points to the side chain of E665 in the stalk region of ADAM10. (B) Close up view of the base (i.e. the presumed membrane proximal face) of the complex shown in (A). In the upper image of (B) a polar contact is indicated (yellow dotted line highlighted by yellow arrow) between the side chain of E665 in ADAM10 and S203 (main chain indicated) of α-toxin. Side chains of R200 and W179 of α-toxin are on the right hand. Lower image in (B) shows result for in silico mutant E665Q.

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: In Silico, Mutagenesis

Journal: bioRxiv

Article Title: Glutamine 666 renders murine ADAM10 an inefficient S. aureus α-toxin receptor

doi: 10.1101/2022.05.11.491455

Figure Lengend Snippet: Phylogenetic tree based on multiple sequence alignment of ADAM10 protein sequences (supplementary information) of different species; carriers of glutamine Q666 (e.g. mouse, or corresponding position in orthologues) highlighted in magenta.

Article Snippet: AB19026 ADAM10 rabbit polyclonal antibody (Immunogen: hADAM10 peptide 732-748) was purchased from Merck; anti-ADAM10 IC1427F was from R&D; anti-HA-Tag 6E2, a mouse mAb, was from CellSignaling (#2367).

Techniques: Sequencing

Journal: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet: ( A ) Venn diagram showing common and exclusive proteins between Integrin adhesion complexes (blue circle) and tunneling nanotubes (TNTs) (yellow circle). The percentages refer to total proteins. ( B ) Venn diagram showing common and exclusive proteins between consensus adhesome (blue circle) and TNTs (yellow circle). ( C ) Representative immunofluorescence pictures showing expression of Integrin b1 and CD151 in TNTs of U2OS and SH-SY5Y as indicated on the left. Each picture is one upper slice of the stack, TNTs are further characterized by actin presence (actin chromobody-GFP, first lane) or wheat germ agglutinin (WGA) labeling. The yellow arrowheads point to TNTs, scale bars are 10 μm. ( D ) Representative immunofluorescence pictures showing labeling of Vinculin and Paxillin in U2OS cells cultured in complete medium or serum-free medium for 24 hr before fixation, as indicated on the left. For each labeling, the bottom slice of the stack is shown on the bottom row (z number), upper slice shows a TNT, pointed with the yellow arrowhead. Red is phalloidin staining, blue is DAPI in merge pictures; scale bars are 10 μm. ( E ) Representative immunofluorescence pictures showing labeling of Vinculin and Paxillin in SY-SY5Y cells, as in D. ( F ) Representative immunofluorescence pictures showing labeling of GM130 in U2OS cells (left, complete or serum-free medium), and SH-SY5Y cells. The yellow arrowheads point to TNTs, scale bars are 10 μm. ( G ) Expression of TM proteins in U2OS whole cell extracts (WCE), which are not in TNTome. WB from wild-type (WT) or GFP-CD9 expressing cells, incubated with the following antibodies as indicated on the left: Integrin b4 (Int b4), a4 (Int a4), EGFR and Connexin 43 (Cx43). Annexin A2 (ANXA2) is used as a loading control. WCE from all cell lines have been tested three times, two WCE are shown. ( H ) Comparative expression of proteins in WCE and TNTs from various U2OS cell lines. Left, CD9, GFP-CD9, and ADAM10 are compared in WT and GFP-CD9 expressing U2OS cells (using non-reducing gels). Right, Int b1 and ANXA2 are compared in H2B-GFP and Actin chromobodies-expressing cells (gels in reducing conditions). Figure 2—figure supplement 1—source data 1. Uncropped and labeled western blots (WBs) for . Figure 2—figure supplement 1—source data 2. Raw unedited western blots (WBs) for . Figure 2—figure supplement 1—source data 3. Uncropped and labeled western blots (WBs) for . Figure 2—figure supplement 1—source data 4. Raw unedited western blots (WBs) for .

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Immunofluorescence, Expressing, Labeling, Cell Culture, Staining, Incubation, Control, Western Blot

Journal: eLife

Article Title: Proteomic landscape of tunneling nanotubes reveals CD9 and CD81 tetraspanins as key regulators

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-ADAM10 11G2 , , Diaclone: #857.800.000 , WB (1/1000).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Marker, Sequencing, Purification, Transduction, Control, Software, Staining

Journal: 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: FIGURE 1. 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 2. The large extracellular loop (LEL) of Tspan14 is the region that interacts with ADAM10 and is required for ADAM10 maturation. A, sche- matic of Tspan14 and CD9 chimeras. The large extracellular loop (LEL) and variable(var)regionofCD9(black)andTspan14(gray)wereinterchanged;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 bufferandimmunoprecipitatedwithananti-FLAGantibody.Immunoprecipi- tated 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 com- pared 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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, Quantitation Assay, Immunoprecipitation, Imaging, Transformation Assay, Comparison

Journal: 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: FIGURE4.AllTspan14-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 Fig. 2A) 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Staining, Confocal Microscopy

Journal: 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: FIGURE 3. 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 Fig. 2A) 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 fluores- cent 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 5. 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 7. 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 exceptananti-calnexinantibodywasusedinsteadofWGAtodefinethelimitsof 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-ADAM10orHA-ADAM1017DCSsignallocalizedintheER.Dataarerepresen- tativeofthreeindependentexperimentsandatleast15fieldsofview.Atwo-way ANOVA statistical analysis was performed with a Bonferroni’s multiple compari- sonstest(ns,non-significant,****,p0.0001).C,HEK-293Tcellsweremocktrans- fected(),ortransfectedwithHA-taggedmouseADAM10wild-typeorADAM10 17DCS. Cells were surface biotinylated, lysed, and immunoprecipitated with an anti-HAantibody.Immunoprecipitateswerestainedwithneutravidin(toppanel) or an anti-HA antibody (bottom panel). Whole cell lysates were stained with an anti-HA antibody (middle panel).

Article Snippet: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 6. 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 nega- tive control (). Lysates were extracted in 1% digitonin lysis buffer and pro- teins 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 immunoprecipi- tated Tspan14, which was arbitrarily set at 100. Data were normalized by log transformationandstatisticallyanalyzedusingaone-wayANOVAwithaDun- nett’s multiple comparison test compared with the mock. All TspanC8s boundsignificantlytoADAM1710DCS(p0.0001).Errorbarsrepresentstan- dard 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 arbi- trarily set at 100. Error bars represent standard error of the mean from three experiments.

Article Snippet: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

Techniques: Transfection, Expressing, Construct, Control, Lysis, Immunoprecipitation, Comparison

Journal: 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: FIGURE 8. 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 9. 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 blottedwithanti-HAtagantibody(toppanel)oranti-FLAGantibody(lowerpanel).Wholecelllysateswereprobedwiththeanti-Myctagantibody(middlepanel). B, data from panel A (upper panel) were quantitated and presented as the amount of immunoprecipitated ADAM10DCS relative to the Tspan14 immunopre- cipitation, 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. AllTspanC8sboundsignificantlytoADAM10DCS(p0.0001).EandF,theseexperimentswerecarriedoutasforpanelsAandBexceptusingHA-taggedhuman ADAM10S (****, p 0.0001; **, p 0.01; *, p 0.05).

Article Snippet: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), 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: 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: FIGURE 10. Evidence that different TspanC8s interact with ADAM10 by distinct mechanisms. A, comparison of TspanC8 co-immunoprecipita- tions with ADAM10 truncation constructs. Quantitation of the co-immu- noprecipitations of ADAM10DCS, ADAM10CS, and ADAM10S with each tetraspanin from Fig. 9 were compared. Values were normalized using Tspan14 data from Fig. 8. All data were relative to the co-immunoprecipi- tation 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 repre- sent 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, respec- tively; for the latter, Tspan14 is depicted as an example.

Article Snippet: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

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

Journal: 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: FIGURE11.DifferentialeffectsofTspanC8sonADAM10substratecleavage:Tspan15promotescleavageofN-cadherinandTspan14reducescleavage ofGPVI.A,HEK-293Tcellsweremocktransfected()ortransfectedwithFLAG-taggedmouseTspanC8s.Thecellswerelysedin1%TritonX-100lysisbufferand 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,datafromA(upperpanel)werequantitatedandthelower,cleavedbandgivenasapercentageofthetotal(upperandlowerbandcombined).Datawere 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 FcR 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 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: Antibodies—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 antihuman ADAM10, and goat anti-mouse ADAM10 (R&D Systems), mouse anti-CD9 (C9-BB) (14), mouse anti-human N-cadherin (BD Biosciences), rabbit anti-GFP (ab290), and mouse anti-human calnexin (AF18) (Abcam).

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