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: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: Several TspanC8 tetraspanins interact with ADAM10 in a digitonin-resistant manner. (A) HCT116 cells were lysed in the presence of Brij 97 or digitonin before immunoprecipitation with the indicated mAb. The composition of the complexes was analyzed by Western blot. This experiment is representative of several experiments performed with various cell lines. Int.α2: integrin α2. (B) PC3 cells were transiently transfected with V5-tagged Tspan1, Tspan5, or Tspan9 and interaction with ADAM10 was analyzed by coimmunoprecipitation after digitonin lysis and Western blot using mAb to ADAM10 (top) or V5 tag (bottom). A10: ADAM10. (C) PC3 cells were transiently transfected with the indicated GFP-tagged tetraspanins and interaction with ADAM10 was analyzed by coimmunoprecipitation after digitonin lysis and Western blot using mAb to ADAM10 (top) or GFP (bottom). (D) Analysis of ADAM10 interaction with nonpalmitoylatable Tspan5 (Tspan5-plm) after digitonin lysis. (E) HEK 293 cells were transfected with HA-tagged ADAM10 and plasmids encoding either GFP or the indicated GFP-tagged tetraspanins. After cross-linking with DSP, the different tetraspanins were immunoprecipitated using the anti-GFP antibody. The samples were electrophoresed under reducing conditions to break the cross-linker, and the presence of ADAM10 cross-linked to the tetraspanins was detected by Western blot using an anti-HA antibody (top). The efficient immunoprecipitation of the different tetraspanins is controlled by immunoblotting the samples using an anti-GFP antibody (bottom). (F) S2 cells were transiently transfected with plasmids encoding Kuz-myc and GFP-Tsp3A, alone or in combination, and lysed using digitonin. The interaction between Kuz and Tsp3A was analyzed by immunoprecipitation and Western blotting using anti-Myc (Kuz) and anti-GFP (Tsp3A) antibodies. All experiments were performed at least twice.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Immunoprecipitation, Western Blot, Transfection, Lysis

Journal: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: ADAM10 localization in the ER correlates with low TspanC8 levels. (A) Flow cytometric analysis of the surface expression of ADAM10 in HeLa, PC3, and HCT116 cells. (B) RT-qPCR analysis of ADAM10 mRNA levels in HeLa, PC3, and HCT116 cells. The figure shows the mean ± SD of three independent experiments performed in triplicate. (C) Surface and intracellular expression of ADAM10 in HeLa, PC3, and HCT116 cells. Cells grown on coverslips were fixed with paraformaldehyde and permeabilized or not with 0.1% Triton X-100 before staining. The acquisition settings were the same for all conditions. This experiment was repeated at least three times. Bar, 10 µm. (D) Confocal microscope analysis of ADAM10 (red) and PDI (green) distribution in HeLa cells permeabilized with 0.1% Triton X-100. This experiment was repeated at least three times. Bar, 10 µm. (E) RT-qPCR analysis of TspanC8 mRNA levels in HeLa, PC3, and HCT116 cells. The figure shows the mean ± SD of three independent experiments performed in triplicate.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Expressing, Quantitative RT-PCR, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: Expression of TspanC8 in HeLa cells increases the expression of surface and mature ADAM10. (A) Flow cytometry analysis of the surface expression of ADAM10 in HeLa cells transiently transfected with the indicated GFP-tagged tetraspanins. (B) Western blot analysis of mature ADAM10 and GFP-tagged tetraspanins in transfected HeLa cells. A nonspecific band is indicated by an asterisk. (C) After biotin labeling of surface proteins, HeLa cells stably expressing or not GFP-tagged Tspan5, Tspan14, and Tspan15 were lysed and the interaction of ADAM10 with the transfected tetraspanins was analyzed by coimmunoprecipitation and Western blot. The major 68-kD band revealed by the anti-ADAM10 mAb perfectly overlapped with the band labeled ADAM10 in the top panel. A nonspecific band is indicated by an asterisk. All experiments were performed at least twice with similar outcome.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Expressing, Flow Cytometry, Transfection, Western Blot, Labeling, Stable Transfection

Journal: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: TspanC8 tetraspanins regulate the subcellular distribution of ADAM10/Kuzbanian in human and Drosophila cultured cells. (A) Confocal microscopy analysis of ADAM10 localization (red) in permeabilized HeLa cells transiently transfected or not with different GFP-tagged tetraspanins (green). Bar, 10 µm. (B) Regulation of the plasma membrane localization of Kuz by Tsp86D and Tsp3A. Transiently transfected S2 cells were scored blind for the localization of Myc-tagged Kuz at the plasma membrane (as in B′) or predominantly in the cytoplasm (as in B′′). Co-transfection of GFP-Tsp86D and GFP-Tsp3A increased the percentage of cells with Kuz at the plasma membrane (B). n is the number of cells that were scored. All experiments were performed at least twice with similar outcome.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Cell Culture, Confocal Microscopy, Transfection, Clinical Proteomics, Membrane, Cotransfection

Journal: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: Endogeneous TspanC8 tetraspanins are required for ADAM10 exit from the ER. (A) Analysis of Tspan5, Tspan14, or Tspan15 expression using RT-qPCR 24 h after the initiation of Tspan14 or Tspan15 silencing in HCT116 and PC3 cells, respectively. The figure shows the mean ± SD of three independent experiments in triplicate. (B) Flow cytometric analysis of the surface expression of ADAM10 in HCT116 or PC3 cells 2 d after the initiation of Tspan14 or Tspan15 silencing, respectively. (C) Confocal microscope analysis of the distribution of ADAM10 (red) and PDI (green) in Triton X-100 permeabilized HCT116 or PC3 cells after 2 d of silencing with a control siRNA or siRNA targeting Tspan14 (HCT116) or Tspan15 (PC3). Bar, 10 µm. All experiments were performed at least twice with similar outcome.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Expressing, Quantitative RT-PCR, Microscopy, Control

Journal: The Journal of Cell Biology

Article Title: TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

doi: 10.1083/jcb.201201133

Figure Lengend Snippet: Tspan5 and Tspan14 regulate ligand-induced Notch activation. (A) Notch activity measured using a CSL reporter luciferase assay, of HeLa cells stably expressing or not Tspan5, Tspan14, or Tspan15. Notch was activated by incubation with OP9-DLL1 cells. The figure shows the mean ± SD of four independent experiments in duplicate. (B) U2OS-N1 cells were treated with the indicated siRNA before analysis of Notch activity produced by incubation with OP9 or OP9-DLL1 cells. The figure shows the mean ± SD of three independent experiments performed in duplicate. (C) Flow cytometric analysis of the surface expression of ADAM10 in U2OS-N1 cells treated with the indicated siRNA. The figure shows the mean ± SD of three independent experiments. (D) Flow cytometric analysis of the surface expression of Notch1 in U2OS-N1 cells treated with a control siRNA or siRNa directed to both Tspan5 and Tspan14. The figure shows the mean ± SD of three independent experiments. (E) U2OS-N1 cells were treated with the indicated siRNA and transfected with NICD and Notch1-ΔE constructs. Notch activity was determined using the luciferase assay. The figure shows the mean ± SD of three independent experiments performed in duplicate. **, P < 0.01; *, P < 0.05 as compared with control cells.

Article Snippet: The mouse mAb anti–human ADAM10 11G2 was described in ; the mouse mAb anti–human Notch1 and anti–human ADAM17 were from R&D Systems and the anti–human PDI from Abcam.

Techniques: Activation Assay, Activity Assay, Luciferase, Stable Transfection, Expressing, Incubation, Produced, Control, Transfection, Construct

Journal: bioRxiv

Article Title: Nanobody-targeted conditional antimicrobial therapeutics

doi: 10.1101/2024.02.20.580917

Figure Lengend Snippet: (A) Design of a conditional antimicrobial therapeutic for delivery of POL. (B) Characterization of POL conjugate VHH16-(ABD) 2 -(EEG) 6 -S17-POL (Left: SDS-PAGE; Coomassie blue staining, Right: Analysis by MALDI-ToF MS reported as mass-to-charge ratio m/z). (C) In vitro cleavage assay of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 by ADAM10 detected via Cy7 fluorescence using an Odyssey CLx imager. (D) In vitro evaluation of masking of antimicrobial activity by VHH16-(ABD) 2 -(EEG) 6 -S17-POL in a microdilution assay on PAO1. Bacterial viabilities were measured based on OD600 absorbance measurements normalized to the untreated control. (E) Experimental timeline and workflow for in vivo evaluation of biodistribution and activation of POL-Cy7 conjugates. (F) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % injected dose (ID)/gram (g). Panels D and F were plotted as mean ± standard deviation (SD). (n = 3). Panel F was analyzed with One-way ANOVA with Tukey post hoc tests. Selected comparisons between POL-Cy7 and released POL-Cy7 from the S17 conjugates were shown in pink. * denotes statistical significance ( P < 0.05). Panel E was partly created with BioRender.com.

Article Snippet: Aliquots of the single cell suspensions (5 x 10 6 cells per aliquot) were first stained with Zombie AquaTM fixable viability kit (1:500 dilution in PBS) and Fc-blocked with TruStain FcX TM (anti-mouse CD16/32) antibody (BioLegend, CA, U.S.A.) (1:20 dilution in PBS (1% BSA)) before further staining with Alexa Fluor TM 488 anti-mouse Ly6G antibody (Clone 1A8) (BioLegend, CA, U.S.A.) (1:100 dilution in PBS (1% BSA)) and DyLight TM 550 anti-mouse ADAM10 antibody (Clone RM0146-7H12) (Novus Biologicals, CO, U.S.A.) (1:100 dilution in PBS (1% BSA)).

Techniques: SDS Page, Staining, In Vitro, Cleavage Assay, Fluorescence, Activity Assay, Microdilution Assay, Control, In Vivo, Activation Assay, Infection, Injection, Standard Deviation

Journal: bioRxiv

Article Title: Nanobody-targeted conditional antimicrobial therapeutics

doi: 10.1101/2024.02.20.580917

Figure Lengend Snippet: (A) Experimental timeline for in vivo evaluation of biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -Sx-POL-Cy7 with different cleavable linkers (Sx). (B) Quantification of total and activated fractions of the POL-Cy7 conjugates in PAO1-infected lungs presented as % ID/g. (C) Experimental timeline for in vivo evaluation of biodistribution and activation of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 using intratracheal pre-treatment with either an excess of VHH16-(ABD) 2 -(EEG) 6 or of the ADAM10-selective inhibitor GI254023X. (D) Quantification of total and activated fractions of VHH16-(ABD) 2 -(EEG) 6 -S17-POL-Cy7 in PAO1-infected lungs presented as % ID/g. (E) Experimental timeline for analysis by flow cytometry of VHH16-(ABD) 2 -(EEG) 6 -NC-SulfoCy5 accumulation in different cell populations of PAO1-infected lungs. (F) A representative dot plot of the lung cell populations based on in vivo accumulated VHH16 and ex vivo stained Ly6G using an anti-Ly6G monoclonal antibody. Gates were set based on the intensity of VHH16 (+/-) and Ly6G (hi/int/neg). (G) Quantification of each cell population presented as percentage of the total live cell population. (H) Quantification of ADAM10 in each cell population based on ex vivo staining with an anti-ADAM10 monoclonal antibody, presented as median fluorescence intensity (MFI). Panels B, D, G, and H were plotted as mean ± SD. (n = 3). Panels B, D, and H were analyzed with One-way ANOVA with Tukey post hoc tests. Selected comparisons between released POL-Cy7 from the S17 conjugate and the other conjugates were shown in pink. * denotes statistical significance ( P < 0.05).

Article Snippet: Aliquots of the single cell suspensions (5 x 10 6 cells per aliquot) were first stained with Zombie AquaTM fixable viability kit (1:500 dilution in PBS) and Fc-blocked with TruStain FcX TM (anti-mouse CD16/32) antibody (BioLegend, CA, U.S.A.) (1:20 dilution in PBS (1% BSA)) before further staining with Alexa Fluor TM 488 anti-mouse Ly6G antibody (Clone 1A8) (BioLegend, CA, U.S.A.) (1:100 dilution in PBS (1% BSA)) and DyLight TM 550 anti-mouse ADAM10 antibody (Clone RM0146-7H12) (Novus Biologicals, CO, U.S.A.) (1:100 dilution in PBS (1% BSA)).

Techniques: In Vivo, Activation Assay, Infection, Flow Cytometry, Ex Vivo, Staining, Fluorescence

Journal: bioRxiv

Article Title: Nanobody-targeted conditional antimicrobial therapeutics

doi: 10.1101/2024.02.20.580917

Figure Lengend Snippet: (A) Design of conditional antimicrobial therapeutic for delivery of PNT4. (B) In vitro cleavage assay of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7 by ADAM10 detected via sulfo-Cy7 fluorescence using an Odyssey CLx imager. (C) In vitro evaluation of antimicrobial activity masking of VHH16-ABD-(EEG) 6 -S17-PNT4 via microdilution assay on PAO1. Bacteria viabilities were measured based on OD600 absorbance normalized to the untreated control. (D) Experimental timeline for in vivo evaluation of biodistribution and activation of VHH16-ABD-(EEG) 6 -S17-PNT4-sulfo-Cy7. Quantification of total and activated fractions of the PNT4-sulfo-Cy7 in (E) PAO1-infected lungs, (F) liver, and (G) kidneys presented as % ID/g. (H) Experimental timeline for in vivo evaluation of therapeutic efficacy of VHH16-ABD-(EEG) 6 -S17-PNT4. (I) Quantification of bacterial burden from the treated lungs presented as log(cfu/g). Dotted line denotes limit of detection. Panels C, E-G, and I were plotted as mean ± SD. (n = 3 for panels C and E-G). (n = 5 for panel I). Panels E-G, and I were analyzed with One-way ANOVA with Tukey post hoc tests. Selected comparisons between PNT4-sulfo-Cy7 and released PNT4-sulfo-Cy7 from the conditional therapeutics were shown in pink. * denotes statistical significance ( P < 0.05). The evaluation of efficacy was confirmed in two independent studies with similar results.

Article Snippet: Aliquots of the single cell suspensions (5 x 10 6 cells per aliquot) were first stained with Zombie AquaTM fixable viability kit (1:500 dilution in PBS) and Fc-blocked with TruStain FcX TM (anti-mouse CD16/32) antibody (BioLegend, CA, U.S.A.) (1:20 dilution in PBS (1% BSA)) before further staining with Alexa Fluor TM 488 anti-mouse Ly6G antibody (Clone 1A8) (BioLegend, CA, U.S.A.) (1:100 dilution in PBS (1% BSA)) and DyLight TM 550 anti-mouse ADAM10 antibody (Clone RM0146-7H12) (Novus Biologicals, CO, U.S.A.) (1:100 dilution in PBS (1% BSA)).

Techniques: In Vitro, Cleavage Assay, Fluorescence, Activity Assay, Microdilution Assay, Bacteria, Control, In Vivo, Activation Assay, Infection, Drug discovery

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

doi: 10.1007/s00018-012-0960-2

Figure Lengend Snippet: Activity analysis of ADAM10. a-I: murine (m)ADAM10 expressed or coexpressed with TSPAN15-myc in Cos7 cells. Activity of ADAM10 was studied through the analysis of ADAM10-specific shedding events using immunoblot detection of N-cadherin [full-length (Fl) N-cadherin (130 kDa), C-terminal fragment (CTF) N-cadherin (37 kDa)] applying a C-terminal-specific N-cadherin antibody. II: EGFP and TSPAN15-myc were transiently expressed in Cos7 cells, and the processing of N-cadherin was analyzed by Western blot. b N2A cells were transfected with pcDNA3.1 or TSPAN15-myc and APP/pcDNA3.1 or APP/TSPAN15-myc. Processing of APP was analyzed using a C-terminal-specific anti-APP antibody. Actin served as protein loading control. c Cell culture supernatants of N2A cells transfected with EGFP or TSPAN15-myc and sAPPalpha content were determined using sandwich ELISAs. d HEK293 cells were transfected with human TSPAN15-EGFP or vector control, and endogenous APP processing was analyzed. APP (FL) APP full-length protein; APP (CTF) APP C-terminal C83 fragment; overex overexposed image of the upper panel. ADAM10, TSPAN15 and actin expression analysis was included

Article Snippet: The following antibodies were used: B42.1, polyclonal rabbit anti-mouse ADAM10 serum (gift of Wim Annaert, Leuven), anti-ADAM10 antibody raised against a peptide corresponding to the C-terminus of murine ADAM10 (used for immunoprecipitation and pulse-chase experiments), anti-actin (IB, Sigma-Aldrich, Hamburg, Germany), MAB946 anti-ADAM10 ectodomain (FACS and IF, R&D Systems, Wiesbaden, Germany), anti-human ADAM10 antibody (11G2) (Gen-Probe, Wiesbaden, Germany), anti-KDEL antibody (Enzo Life Science, Lörrach, Germany), anti-PDI antibody (Santa Cruz Biotechnology, Santa Cruz, USA), 9B11 anti-myc (IP, IB and IF, Cell Signaling Technology, Frankfurt am Main, Germany), B63.3 polyclonal rabbit anti-mouse APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), H68.4 anti-transferrin receptor (IB, Life Technologies GmbH, Darmstadt, Germany); FL-335 anti-GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany) and anti N-cadherin (BD Transduction Laboratories, Heidelberg, Germany).

Techniques: Activity Assay, Western Blot, Transfection, Cell Culture, Plasmid Preparation, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

doi: 10.1007/s00018-012-0960-2

Figure Lengend Snippet: TSPAN15-myc influences ADAM10 localization. a-I, II: Murine ADAM10 and ADAM10/TSPAN15-myc expressed in Cos7 cells. III, IV: Murine ADAM17 and ADAM17/TSPAN15-myc expressed in Cos7 cells. Confocal immunofluorescence pictures were taken using an anti-KDEL antibody, an anti-ADAM10 antibody, and an anti-ADAM17 antibody, respectively. Scale bar 100 μm. V, VI: SHSY cells were transiently transfected with a C-terminal EGFP-tagged variant of human TSPAN15. Endogenous ADAM10 was stained using an anti-ADAM10 antibody (11G2), and an anti-PDI antibody was used as ER marker. Asterisks mark untransfected cells. Scale bar 10 μm. b N2A cells were biotinylated after transfection with either EGFP or murine TSPAN15-myc. Following cell lysis total protein samples were taken, and after precipitation of biotin-labeled proteins immunoblotting of total lysates and bound fractions was performed. TSPAN15-myc and ADAM10 were detected. The detection of the transferrin receptor (TFR) was included as a control for biotinylated surface proteins, and antibodies against the intracellular glycerin-aldehyd-3-phosphate dehydrogenase (GAPDH) were used as a negative control. c-I: N2A cells were transfected with ADAM10, TSPAN15-myc and ADAM10/TSPAN15-myc; ADAM10 cell surface expression was determined through FACS analysis using an N-terminal anti-ADAM10 antibody. RFUs were determined. Statistical significance was determined using Student’s t test; values are provided as highly significant (**p < 0.01). Abbreviation: relative fluorescence units (RFU). II: Representative overlay of the FACS analysis performed

Article Snippet: The following antibodies were used: B42.1, polyclonal rabbit anti-mouse ADAM10 serum (gift of Wim Annaert, Leuven), anti-ADAM10 antibody raised against a peptide corresponding to the C-terminus of murine ADAM10 (used for immunoprecipitation and pulse-chase experiments), anti-actin (IB, Sigma-Aldrich, Hamburg, Germany), MAB946 anti-ADAM10 ectodomain (FACS and IF, R&D Systems, Wiesbaden, Germany), anti-human ADAM10 antibody (11G2) (Gen-Probe, Wiesbaden, Germany), anti-KDEL antibody (Enzo Life Science, Lörrach, Germany), anti-PDI antibody (Santa Cruz Biotechnology, Santa Cruz, USA), 9B11 anti-myc (IP, IB and IF, Cell Signaling Technology, Frankfurt am Main, Germany), B63.3 polyclonal rabbit anti-mouse APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), H68.4 anti-transferrin receptor (IB, Life Technologies GmbH, Darmstadt, Germany); FL-335 anti-GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany) and anti N-cadherin (BD Transduction Laboratories, Heidelberg, Germany).

Techniques: Immunofluorescence, Transfection, Variant Assay, Staining, Marker, Lysis, Labeling, Western Blot, Negative Control, Expressing, Fluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

doi: 10.1007/s00018-012-0960-2

Figure Lengend Snippet: Early interaction of TSPAN15 with ADAM10. a Murine ADAM10 was either expressed with TSPAN15-myc or TSPAN15-ER-myc. Cells were lysed, and myc-tagged proteins were precipitated using an anti-myc antibody. After immunoblotting coprecipitated ADAM10 was detected using a C-terminal-specific antibody (B42.1). b-I, II: Cos7 cells were transfected with TSPAN15-ER-myc or TSPAN15-ER-myc/ADAM10. Confocal immunofluorescence pictures were taken using an anti-myc antibody, an anti-KDEL antibody and an N-terminal-specific ADAM10 antibody and adequate secondary antibody pairs. Scale bar 100 μm. c N2A cells were transfected with EGFP or TSPAN15-myc, pulsed for 1 h with 35S methionine/cysteine and chased for 0, 2, 6, 18 and 30 h. After cell lysis equal amounts of protein were used, and ADAM10 was precipitated using a C-terminal-specific anti-ADAM10 antibody, subjected to SDS-PAGE and analyzed by fluorographics. TSPAN15-myc expression was analyzed by Western blot, and actin served as protein-loading control for the lysates

Article Snippet: The following antibodies were used: B42.1, polyclonal rabbit anti-mouse ADAM10 serum (gift of Wim Annaert, Leuven), anti-ADAM10 antibody raised against a peptide corresponding to the C-terminus of murine ADAM10 (used for immunoprecipitation and pulse-chase experiments), anti-actin (IB, Sigma-Aldrich, Hamburg, Germany), MAB946 anti-ADAM10 ectodomain (FACS and IF, R&D Systems, Wiesbaden, Germany), anti-human ADAM10 antibody (11G2) (Gen-Probe, Wiesbaden, Germany), anti-KDEL antibody (Enzo Life Science, Lörrach, Germany), anti-PDI antibody (Santa Cruz Biotechnology, Santa Cruz, USA), 9B11 anti-myc (IP, IB and IF, Cell Signaling Technology, Frankfurt am Main, Germany), B63.3 polyclonal rabbit anti-mouse APP C-terminus (IB, a kind gift of Wim Annaert, Leuven, Belgium), H68.4 anti-transferrin receptor (IB, Life Technologies GmbH, Darmstadt, Germany); FL-335 anti-GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany) and anti N-cadherin (BD Transduction Laboratories, Heidelberg, Germany).

Techniques: Western Blot, Transfection, Immunofluorescence, Lysis, SDS Page, Expressing