Journal: Frontiers in Cell and Developmental Biology

Article Title: NLGN3 Upregulates Expression of ADAM10 to Promote the Cleavage of NLGN3 via Activating the LYN Pathway in Human Gliomas

doi: 10.3389/fcell.2021.662763

Figure Lengend Snippet: NLGN3 activated the LYN pathway to upregulate ADAM10, which in turn promoted cleavage of NLGN3. (A) The NLGN3 plasmid carrying the Flag fragment was transfected into U251 cells with an empty vector as the control. Western blot was performed to detect the Flag label. SS = signal sequence; M = maker. (B) The level of phosphorylation of AKT and LYN was detected after treatment with a NLGN3 plasmid, with an empty vector as the control. WT = wild type. (C) U251 cells were transfected with LYN or treated with bafetinib along with NLGN3 plasmid transfection, with an empty vector or DMSO as the control. Western blot was performed to detect the Flag label. WCL = whole cell lysates. (D) qPCR was performed to detect the expression of ADAM10 in U251 cells. (E) Western blot was performed to detect the expression of ADAM10 in U251 cells. ADAM10 expression was upregulated by LYN and inhibited by bafetinib. (F) Compared with the control cells, LYN overexpression increased the level of cleaved NLGN3, whereas decreased cleaved NLGN3 was observed after treatment with the ADAM10 inhibitor compared with LYN overexpression cells. (G) Overexpression of ADAM10 increased cleaved NLGN3 levels, but there was no significant change of cleaved NLGN3 levels after treatment with LYN inhibitors compared with ADAM10 overexpression group. * p < 0.05; ** p < 0.01.

Article Snippet: The ADAM10 primer (F: 5′-ATGGTGTTGCTGAGAGTGTT-3′; R: 5′-GACTGCTCTTTTGGCACGC-3′) was purchased from Ribobio Co., Ltd. (Guangzhou, China).

Techniques: Plasmid Preparation, Transfection, Western Blot, Sequencing, Expressing, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: NLGN3 Upregulates Expression of ADAM10 to Promote the Cleavage of NLGN3 via Activating the LYN Pathway in Human Gliomas

doi: 10.3389/fcell.2021.662763

Figure Lengend Snippet: ADAM10 promoted the migration and invasion of glioma cells. (A) Correlation analysis between expression of ADAM10 and NLGN3 in glioma and normal brain tissue was performed using GEPIA. (B) Correlation analysis between expression of ADAM10 and LYN in glioma and normal brain tissue was performed using GEPIA. (C) The expression of ADAM10 in LGG and GBM was analyzed on GEPIA. (D) U87 cells were treated with ADAM10 inhibiter, GI254023X (10 μM), and CCK-8 assay was performed to detect the proliferation of each group of cells. (E,F) ADAM10 inhibiter, GI254023X, suppressed the migration of U87 cells. (G,H) GI254023X inhibited the invasion of U87 and U251 cells. (I) The relationship between ADAM10 and prognosis of patients with LGG and GBM was analyzed on GEPIA. * p < 0.05.

Article Snippet: The ADAM10 primer (F: 5′-ATGGTGTTGCTGAGAGTGTT-3′; R: 5′-GACTGCTCTTTTGGCACGC-3′) was purchased from Ribobio Co., Ltd. (Guangzhou, China).

Techniques: Migration, Expressing, CCK-8 Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: NLGN3 Upregulates Expression of ADAM10 to Promote the Cleavage of NLGN3 via Activating the LYN Pathway in Human Gliomas

doi: 10.3389/fcell.2021.662763

Figure Lengend Snippet: Schematic illustration of the positive feedback circle, s-NLGN3/LYN/ADAM10. Secreted NLGN3 derived from neurons and glioma cells activates LYN and then up-regulates ADAM10 expression, which can cleave NLGN3 to promote its secretion.

Article Snippet: The ADAM10 primer (F: 5′-ATGGTGTTGCTGAGAGTGTT-3′; R: 5′-GACTGCTCTTTTGGCACGC-3′) was purchased from Ribobio Co., Ltd. (Guangzhou, China).

Techniques: Derivative Assay, 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, a new ADAM10 interaction partner. a-I, II: Split-ubiquitin yeast two-hybrid system (modified from [50]). ADAM10 C-terminally fused to the C-terminal part of ubiquitin (Cub) and an artificial transcription factor (LexA-VP16) coexpressed in yeast together with a N-terminal NubG-tagged murine brain library. The close proximity between the ADAM10 bait protein and an interaction partner leads to the reconstitution of “split ubiquitin” to ubiquitin, which is recognized by cellular ubiquitin proteases, which in turn release the artificial transcription factor from the membrane. This enables yeast to grow on selective media [without leucine (-leu), tryptophane (-trp), histidine (-his)] plates. C-terminal part of ubiquitin (Cub, C), N-terminal part of ubiquitin (NubG, N), LexA (L). b ADAM10 bait protein coexpressed with the identified TSPAN15 prey protein and controls in NMY51 yeast. Transfection of both bait and prey protein is verified on Leu/Trp-lacking selective media plates, and interaction of ADAM10 and TSPAN15 is monitored under selective pressure on Leu/Trp/His-lacking media plates in comparison to controls (“+” and “−”). c-I: Mammalian expression constructs of murine ADAM10 and murine TSPAN15-myc were transiently coexpressed in HeLa cells. TSPAN15-myc (35–37 kDa) was precipitated using an anti-myc antibody, and coprecipitation of ADAM10 was detected with an anti-ADAM10 antibody. II: ADAM10 and TSPAN15-myc were transiently coexpressed in HeLa cells, and ADAM10 was precipitated using an anti-ADAM10 antibody. Coprecipitation was analyzed by Western blot using an anti-myc antibody. Single transfections of TSPAN15-myc and ADAM10 served as specificity controls for the antibodies used for immunoprecipitation. III: Quantification of band intensities of pro and mature form of ADAM10 in lysates and CoIP fractions. Ratios of band intensities of pro/(pro + mature) forms of ADAM10 were calculated (%, n = 5). Student’s t test was performed (***p < 0.005). [pro(p) ADAM10, 95 kDa, mature(m) ADAM10, 75 kDa]. Abbreviations: untransfected (Ø), vector (mock) transfected (V), murine ADAM10 (A10), murine TSPAN15-myc (T15), asterisk marks immunoglobulin signals

Article Snippet: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Ubiquitin Proteomics, Modification, Membrane, Transfection, Comparison, Expressing, Construct, Western Blot, Immunoprecipitation, Plasmid Preparation

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 maturation. a Murine ADAM10 transiently coexpressed with either EGFP or murine TSPAN15-myc in Cos7 cells. After immunoblotting ADAM10 was detected using an ADAM10 specific C-terminal antibody (B42.1) and TSPAN15-myc was detected using an anti-myc antibody. b TSPAN15-myc or EGFP was transiently expressed in N2A cells. After lysis proteins were immunoblotted, and endogenous ADAM10 and TSPAN15-myc were detected. c Murine ADAM17 or murine ADAM10 was transiently expressed either with EGFP or TSPAN15-myc in HeLa cells. Presence of ADAM17 [pro(p) ADAM17, closed arrowhead, 120 kDa; putative mature ADAM17, open arrowhead] was analyzed using a C-terminal-specific antibody, and TSPAN15-myc and ADAM10 expressions were monitored as mentioned above. Actin served as protein loading control. Asterisks mark unspecific antibody binding

Article Snippet: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Western Blot, Lysis, Control, Binding Assay

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: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Immunofluorescence, Transfection, Variant Assay, Staining, Marker, Lysis, Labeling, Western Blot, Control, 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: 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: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Activity Assay, Western Blot, Transfection, Control, 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: Knockdown of TSPAN15. a Knockdown of TSPAN15 in N2A cells was verified through qRT-PCR analysis. b N2A cells were transfected with siRNA against TSPAN15 and control siRNA. After lysis and immunoblot analysis, ADAM10 was detected. Actin served as protein-loading control. c-I: Surface expression of ADAM10 was analyzed through FACS analysis using a N-terminal anti ADAM10 antibody. Remaining surface expression of ADAM10 was determined (%). II: Representative overlay of FACS analysis of ADAM10 surface protein after TSPAN15 knockdown

Article Snippet: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Control, Lysis, Western Blot, 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: 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: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

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

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: Model of TSPAN15 function. (1) ADAM10 is synthesized as an inactive precursor (pro-ADAM10) in the ER, and TSPAN15 accelerates its ER exit. (2) After ER exit the immature ADAM10 is activated through removal of the inhibitory prodomain by furin or proprotein convertase (PC7), and is then transported to the plasma membrane together with TSPAN15. (3) At the plasma membrane TSPAN15 facilitates the integration of ADAM10 in the tetraspanin web. (4, 5) ADAM10 is stabilized, and the web composition enables ADAM10 to get access to its substrates and subsequent cleavage events in the juxtamembrane regions of the substrates take place. (6) Afterwards remaining membrane-bound fragments are cleaved through ripping proteases (γ-secretase complex/SPPLs) in the transmembrane regions, thereby liberating soluble intracellular domains (ICD), which might have signaling functions

Article Snippet: ADAM10 (Mm00545742_m1, Applied Biosystems, Foster City, CA, USA), TSPAN15 (Mm01150417_m1, Applied Biosystems) and GAPDH (Mm99999915_g1, Applied Biosystems) expression were determined by real-time PCR analysis of 0.5 μl cDNA on a 7900HT Fast Real-time PCR System (Applied Biosystems) in 10-μl reaction volume in 384-well plates in duplicate.

Techniques: Synthesized, Clinical Proteomics, Membrane

Journal: Biomaterials and Biosystems

Article Title: Engineered extracellular vesicles antagonize SARS-CoV-2 infection by inhibiting mTOR signaling

doi: 10.1016/j.bbiosy.2022.100042

Figure Lengend Snippet: Gene expression assays.

Article Snippet: adam10 , Human , , Hs00153853_m1.

Techniques: Gene Expression

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 3. Anx A1 cleavage is downstream of ADAM10. A, Anx A1 cleavage cannot be blocked by inhibition of elastase or proteinase 3. Jurkat cells were stimulated with 2.5 mM staurosporine in the absence or presence of elastase inhibitor (Ela-I), aprotinin, or Pefabloc for 18 h. Subsequently, cells were lysed, and anx A1 processing was monitored by immunoblot analysis. B, Anx A1 cleavage is mediated by a membrane-resident protease. One microgram of purified recombinant human anx A1 was incubated with the culture supernatant of 4 3 106 secondary necrotic Jurkat cells per milliliter or the membrane fraction of 3 3 107 secondary necrotic Jurkat cells at 37˚C for the indicated times, and anx A1 cleavage was examined by immunoblot analysis. C, Anx A1 cleavage is blocked by o-phenanthroline. Jurkat cells were stimulated as in A in the absence or presence of the metalloproteinase inhibitor o-phenanthroline. Anx A1 processing was detected by immunoblot analysis. Vinculin was used as a loading control. D, Addition of the broad-range matrix metalloproteinase inhibitor GM 6001 blocks anx A1 cleavage. Jurkat cells were stimulated as in A in the presence of 0–100 mM GM 6001. Afterwards, anx A1 cleavage was monitored by immunoblot analysis. PARP was used as a loading and apoptosis/secondary necrosis control. E, Proteolytic processing of anx A1 can be blocked by the ADAM10 inhibitor GI 254023X. Jurkat cells were stimulated as in A in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Subsequently, anti-anx A1 immunoblot analysis was performed with protein extracts as in D. F, Analysis of ADAM10 and ADAM17 knockdown efficiency by qRT-PCR. Knockdown of ADAM10 and ADAM17 expression was carried out by electroporation of Jurkat cells with two different ADAM10- and ADAM17-specific oligonucleotides and a scramble control oligonucleotide as described in Materials and Methods. Total RNA was prepared, reversely transcribed, and the resulting cDNA was used for qRT-PCR as described in Materials and Methods. Relative ADAM10/17 mRNA levels were normalized on the endogenous control ALAS-1, and the ADAM10/17 mRNA level in Jurkat cells that were treated with the scramble control siRNA was set as 100% calibrator. G, Anx A1 cleavage is strongly inhibited in ADAM10 silenced cells. siRNA- mediated knockdown of ADAM10 or ADAM17 expression was performed as in F. Subsequently, cells were stimulated to undergo secondary necrosis as in A, and anx A1 cleavage was monitored by immunoblot analysis. The amount of anx A1 p36 compared with total anx A1 was calculated from integrated pixel intensities, and the inhibition of cleavage is presented as percent of the scramble control. PARP served as a loading and apoptosis/secondary necrosis control.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Inhibition, Western Blot, Membrane, Recombinant, Incubation, Control, Knockdown, Quantitative RT-PCR, Expressing, Electroporation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 4. Anx A1 is directly cleaved by ADAM10 after F7. A, Domain structure of different anx A1 constructs. Arabic numbers depict the amino acid position, and annexin repeats are numbered I–IV. B, Recombinant human anx A1 (aa 1–346) is processed by recombinant human ADAM10. One mi- crogram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng recombinant human ADAM10 ectodomain in the presence or absence of 100 mM of the matrix metalloproteinase inhibitor TAPI-2 at 37˚C for the indicated times. Subsequently, anx A1 cleavage was detected by SDS-PAGE and immunoblot analysis with an anti-anx A1 Ab. C, Recombinant human anx A1 core domain (aa 47–346) is not cleaved by ADAM10. Incubation of anx A1 (aa 47–346) with ADAM10 ectodomain was performed as in B. For immunoblot analysis, a polyclonal anti-anx A1 Ab was used. D, The cleavage site of ADAM10 is located within the unique N-terminal domain of anx A1 (aa 1–46). One microgram of the recombinant human anx A1 N-terminal domain (aa 1–46) was incubated with ADAM10 as in B. Cleavage fragments were separated by SDS-PAGE and visualized by subsequent silver staining. E, Recombinant human anx A1 (aa 1–346) is not processed by recombinant human ADAM17. One microgram of recombinant human anx A1 (aa 1–346) was incubated with 100 ng of recombinant human ADAM17 ectodomain as in B. F, Identification of the ADAM10 cleavage site within the anx A1 N-terminal domain. Recombinant human anx A1 (aa 1–346) was incubated with native or heat-inactivated recombinant human ADAM10 as in A. Subsequently, the reaction mixture was subjected to N-terminal Edman degradation. The N-terminal sequence newly generated by incubation with active ADAM10 was L8

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Construct, Recombinant, Incubation, SDS Page, Western Blot, Silver Staining, Sequencing, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

doi: 10.4049/jimmunol.1004073

Figure Lengend Snippet: FIGURE 6. ADAM10 contributes to the release of “find-me” signals from secondary necrotic cells. A, GM 6001 inhibits the release of monocytic chemoattractants during secondary necrosis. Jurkat, MOLT-4, and THP-1 cells were UV-irradiated and incubated for 12 or 18 h in the presence or absence of the broad-range matrix metalloproteinase inhibitor GM 6001 (100 mM). Cell-free culture supernatants were collected and analyzed for their chemotactic potential as in Fig. 5A. Error bars represent SD of quadruplicates. B, The release of secondary necrotic cell-derived attraction signals is strongly decreased in the presence of the ADAM10-specific inhibitor GI 254023X. Jurkat cells were UV-irradiated and incubated for 18 h in the absence or presence of 10 mM of the ADAM10 inhibitor GI 254023X (GI) or the ADAM10/17 inhibitor GW 280264X (GW). Supernatants were collected and applied to a transmigration assay with THP-1 cells as in Fig. 5A. Error bars represent SD of quadruplicates. C, Evaluation of ADAM10 knockdown efficiency by FACS analysis. Knockdown of ADAM10 expression in Jurkat cells was carried out as in Fig. 3G. On day 5 after the first electroporation, cells were fixed, permeabilized, and stained with anti-ADAM10–PE Ab to detect ADAM10 expression level by FACS analysis or IgG-2b-PE isotype control. Left panel, Representative histograms are shown. Right panel, Median PE fluorescence of the histograms in the left panel. D, The release of monocytic attraction signals is strongly decreased in ADAM10-silenced secondary necrotic cells. ADAM10-silenced cells were induced to undergo secondary necrosis by UV irradiation. Cell culture supernatants were harvested and analyzed for their chemotactic potential in a transmigration assay with THP-1 cells as in Fig. 5A. Mean values + SD of quadruplicates are given.

Article Snippet: Recombinant human MCP-1, SDF-1a, ADAM10, and ADAM17 ectodomains were obtained from R&D Systems.

Techniques: Irradiation, Incubation, Derivative Assay, Transmigration Assay, Knockdown, Expressing, Electroporation, Staining, Control, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 is strongly enriched at presynaptic sites. a Scheme of ADAM10 at the synaptic membrane with indicated C-terminal anti-ADAM10 antibody binding. Nt N-terminus, Ct C-terminus. b – d Validation of the C-terminal ADAM10 antibody. b Immunocytochemistry for ADAM10 in wildtype (+ / + , wt) and knockout (-/-, KO) MEF cells. Representative widefield image ( b 1 ) and quantification ( b 2 ) show strong reduction of ADAM10 immunoreactivity in KO MEF cells. n = 10 (wt), n = 11(KO) images from 1 MEF cell preparation. 2-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. c Immunoblot analysis of ADAM10 wt and KO mouse embryonic fibroblast (MEF) cell lysates, detected with C-terminal ADAM10 antibody, indicates loss of ADAM10 bands corresponding to the precursor and mature form of the protease in KO cells. pA10: precursor of ADAM10; mA10: mature ADAM10. d Immunoblot of P21 A10 cKO and wt cortical extracts shows strong reduction in the ADAM10 signal in the cKO. pA10: precursor of ADAM10; mA10: mature ADAM10. e Representative maximum projections of confocal images of hippocampal primary cultures at div3. Immunostaining for ADAM10 (green), the axonal marker tau (blue), and MAP2 (red) as a dendritic marker. Note the strong enrichment of ADAM10 at the axon and axonal growth cones already in young cultures. f Left: Representative maximum projection of confocal images of a div18 primary rat hippocampal neuron, transfected with a maxGFP cell fill (green) and stained for ADAM10 (red) and the presynaptic vesicle marker synaptophysin (blue) in an axon and at a dendrite. ADAM10 is present at presynaptic boutons. Note that dendritic spines are largely devoid of ADAM10. Right: Line scans of indicated axonal bouton and dendritic spine. g Representative gated STED images of mature rat hippocampal primary neurons (div17) stained for ADAM10 (green), in combination with presynaptic cytomatrix of the active zone (CAZ) protein piccolo (red) and the vesicle marker synaptophysin (blue) or the presynaptic CAZ protein bassoon (blue), and the postsynaptic scaffold shank3 (red). Boxes indicate position of zoom-ins, lines were used for the line profiles shown. Note the localization of ADAM10 on the presynaptic (bassoon, blue) site. Right: Line scans of indicated synapses. See also Figure

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Membrane, Binding Assay, Biomarker Discovery, Immunocytochemistry, Knock-Out, Western Blot, Immunostaining, Marker, Transfection, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 in enriched in vesicles of mossy fiber boutons, that show only minor morphological changes in cKO animals. a DAB staining of ADAM10 in an adult wildtype mouse hippocampus shows strong enrichment of ADAM10 in mossy fibers. DG dentate gyrus, MF Mossy fibers. b High magnification of ADAM10 DAB and control (without primary antibody) staining in MF-CA3 synapses. Note the strong ADAM10 localization to the presynaptic site (pink arrow) and lack of DAB staining at the presynaptic membrane in the control (yellow arrowhead). S: dendritic spine. B: mossy fiber bouton (false coloured in blue). c Immunogold EM of a P21 wildtype mouse with focus on hippocampal mossy fiber boutons. Note that lack of gold particles at the synaptic membrane and that gold particles localize to the outside of vesicles, as the antibody detects ADAM10’s cytosolic C-terminus (see the scheme). d Example of ADAM10 cKO and wt mossy fiber bouton 3D reconstructions from SBEM data. See also Videos S1–S4. e Quantification of MFB volume, surface area and sphericity. n = 19 (wt), n = 23 (cKO) boutons from 3 animals each. 2-tailed unpaired Student’s t-test (volume, surface area) and Mann Whitney U-test (sphericity). Data are represented as mean ± SEM. See also Figure

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Staining, Control, Membrane, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 is required for the expression of presynaptic mossy fiber short-term plasticity which does not depend on the enzymatic activity of the protease. a Image of an acute hippocampal slice with indicated positions for stimulating (MF) and recording (CA3) electrodes. b – d Mossy fiber plasticity of ADAM10 wt and cKO animals. b Paired pulse facilitation ratio at different stimulation frequencies and example traces (average of 5 sweeps, inlet) of evoked fEPSPs at 20 Hz of ADAM10 cKO and wt mice. ADAM10 cKO show impaired facilitation. n = 18 (wt), n = 19 (cKO) slices from 5 mice each. Two-way repeated measures ANOVA. ****p < 0.0001. Data are represented as mean ± SEM. c Example traces of train facilitation at 20 Hz in wt and cKO slices. Application of the group II mGluR agonist DCGIV (1 µM) leads to a loss of response and is used to prove the mossy fiber origin of the detected signal. d Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. ADAM10 cKO slices show an impaired response to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). 2-way repeated measures ANOVA. ****p < 0.0001. n = 18 slices (wt), n = 19 slices (cKO) from 5 mice each. Data are represented as mean ± SEM. e – h MF plasticity of wt animals with or without ADAM10 inhibitor (GI254023X) treatment. e Paired pulse facilitation ratio at different stimulation frequencies and example traces [average of 3 (GI254023X) or 4 (ctr) sweeps, inlet] of evoked fEPSPs at 20 Hz of wt mouse slices upon inhibition of ADAM10 activity. Application of the ADAM10 inhibitor GI254023X does not affect synaptic facilitation. Two-way repeated measures ANOVA. Treatment p = 0.3113. n = 12 slices (control); n = 11 slices (GI254023X) from 3 mice each. Data are represented as mean ± SEM. f Example traces of train facilitation at 20 Hz in wt slices with and without ADAM10 inhibitor (GI254023X). Application of the group II mGluR agonist DCGIV (1 µM) leads to a loss of response and is used to prove the mossy fiber origin of the detected signal. g Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. Treatment with ADAM10 inhibitor does not change the ratio calculated in response to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). Two-way repeated measures ANOVA. p = 0.5764 (10 Hz), p = 0.9124 (20 Hz). n = 12 (control); n = 11 slices (GI254023X) from 3 mice each. Data are represented as mean ± SEM. h Immunoblot analysis of acute hippocampal wt slices untreated or treated with GI254023X confirming that the application of the ADAM10 inhibitor does in fact reduce ADAM10 activity. Note the reduced substrates cleavage (PrPc to shed PrPc, N-cadherin to C-terminal fragment CTF) in the GI254023X group. d di-glycosylated, m mono-glycosylated, u unglycosylated. See also Figure

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Expressing, Activity Assay, Inhibition, Control, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 acts via the syt7 pathway. a Syt7 levels in hippocampus of ADAM10 cKO mice are reduced, while the major mossy fiber calcium buffer calbindin and the vesicle marker VAMP are unchanged. n = 3 acute slice preparations of 3 animals (same slices as in Fig. 4). Unpaired, 2-tailed Student’s test. b Synaptic syt7 associates with ADAM10 in a Ca 2+ -independent manner. Endogenous Co-immunoprecipitations from mouse synaptosomes in presence of 200 µM CaCl 2 or 2 mM EGTA. *Unspecific band. Note the different exposure times for the lower blot. syso: synaptosomes; ctr: control. c Heterologous Co-immunoprecipitations from Neuro-2a cells shows syt7-GFP is in one complex with ADAM10 in both calcium (200 µM) and calcium-free (2mM EGTA) conditions. d , e ADAM10 and syt7 co-localize in primary hippocampal cultures. d Representative maximum projections of STED Xtend superresolution images of mature primary hippocampal cultures stained with a CF488A-pre-labelled ADAM10 antibody (green), syt7 (red) and the presynaptic scaffold bassoon (blue) and line scans. e Proximity ligation assay (PLA) for ADAM10 and syt7 in mature hippocampal cultures showing PLA signals at neuronal processes/axons indicating that both proteins are in close proximity (< 40 nm). Example maximum projection of a spinning disc confocal image showing the PLA signal (blue), cell morphology marker actin (phalloidin, green) and the synaptic marker bassoon (red). See also Figure

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Marker, Control, Staining, Proximity Ligation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet: ADAM10 C-terminus is required for mossy fiber short-term plasticity. a Scheme of tat-peptide in relation to ADAM10 C-terminus. l length, aa amino acids. b Paired pulse facilitation ratio at different stimulation frequencies and example traces (average of 5 sweeps, inlet) of evoked fEPSPs at 20 Hz in hippocampal slices treated with ctr-tat or ADAM10-tat peptide. Application of ADAM10 C-terminus targeted tat-peptide leads to impairment in facilitation. Two-way repeated measures ANOVA. **p = 0.0086. n = 12 slices from 3 mice each. Data are represented as mean ± SEM. c Example traces of train facilitation at 20 Hz in both experimental groups. Application of the group II mGluR agonist DCGIV (1 µM) is used as control for the mossy fiber origin of the detected signals. D Quantification of the ratio calculated from the fEPSP amplitudes measured in response to train facilitation. Impaired response upon ADAM10-tat-peptide treatment to train stimulation at 20 Hz (dark colours) and 10 Hz (light colours). 2-Way repeated measures ANOVA. * p = 0.0294 (10 Hz), * p = 0.0409 (20 Hz). n = 12 slices from 3 mice each. Data are represented as mean ± SEM. e Syt7 levels are not changed in hippocampal slices after treatment with ADAM10-tat peptide compared to control-tat peptide. n = 3 acute slice preparations of 3 animals. Unpaired, 2 -tailed Student’s test. See also Figure

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Non-canonical function of ADAM10 in presynaptic plasticity

doi: 10.1007/s00018-024-05327-8

Figure Lengend Snippet:

Article Snippet: ADAM10- tat-peptide (ADAM10 aa 709–730) (YGRKKRRQRRRPKLPPPKPLPGTLKRRRPPQPI) , Genosphere , N/A.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, Control, In Situ, Knock-Out, Software, Microscopy