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: bioRxiv

Article Title: Tumor necrosis factor is a necroptosis-associated alarmin

doi: 10.1101/2022.08.01.502280

Figure Lengend Snippet: (A) 293T cells stably transduced with MLKL 1-201 were transfected with pRP-TNF or pRP-IL6. The day after cells were treated with 1 μg/ml doxycycline or left untreated for 7 h and LDH and cytokine release were measured. The right panel depicts the cytokine release data as fold change data in relation to the mean value of the data of the untreated cells. (B) Expression of genes of the ADAM family members as from RNA-seq data of WT BLaER1 cells stimulated as indicated. (C) Immunoblotting of ADAM17 and ADAM10 in BLaER1 of the indicated genotypes. (D) IL-6Rα secreted by CASP8 -/- × IL6 -/- and CASP8 -/- × MLKL -/- × IL6 -/- treated with 2 ng/ml LPS for 18 h. Data are depicted as mean ± SEM of 3 independent experiments (A and D). Statistics indicates significance by an unpaired, two-tailed t-test (A) or a two-way ANOVA (D) with a Šidák (D) correction for multiple testing: *** P<0.001, * P<0.05, ns=not significant. Raw data are available in —source data 1 and 2.

Article Snippet: The following antibodies were purchased from commercial suppliers: Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific, Cat# A-11001, RRID:AB_2534069 and Cat# A-11008, RRID:AB_143165), anti-b-actin (C4) HRP (Santa Cruz Biotechnology, Cat# sc-47778 HRP, RRID:AB_271418), anti-Caspase-8 (1C12) (Cell Signaling Technology, Cat# 9746, RRID:AB_2275120), anti-ADAM10, CT (Millipore, Cat# AB19026, RRID:AB_2242320), anti-ADAM17 (D22H4) (Cell Signaling Technology, Cat# 6978, RRID:AB_1082838), anti-IκBα (L35A5) (Cell Signaling Technology, Cat# 4814, RRID:AB_390781), anti-phospho-IkBa (14D4) (Cell Signaling Technology, Cat# 2859, RRID:AB_561111), anti-TNF (D5G9) (Cell Signaling Technology, Cat# 6945, RRID:AB_10859375), CaptureSelect™ Alexa488 anti-C-tag conjugate (Thermo Fisher Scientific, Cat#7213252100), MLKL polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-34733, RRID:AB_2552085), phospho-NF-κB p65 (Ser536) (93H1) rabbit mAb (Cell Signaling Technology, Cat# 3033, RRID:AB_331284), recombinant anti-MLKL (phospho S358) antibody (Abcam, Cat# ab187091, RRID:AB_2619685).

Techniques: Stable Transfection, Transduction, Transfection, Expressing, RNA Sequencing Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Tumor necrosis factor is a necroptosis-associated alarmin

doi: 10.1101/2022.08.01.502280

Figure Lengend Snippet: (A) 293T (control), 293T MLKL 1-201 and ADAM10 -/- × ADAM17 -/- MLKL 1-201 293T were transfected with pLI-C-tag-TNF linker mCherry and pEF-BOS-nBFP and stimulated with 1 μg/ml doxycycline and imaged over time starting 4 h after doxycycline treatment. Exemplary images of the indicated channels 8h30 after doxycycline induction are shown. Bar = 25 μm. (B) Trajectories of TNF mCherry and C-tag TNF of cells treated as in (A). Five representative cells per cell type are shown. (C) One exemplary cell from the 293T MLKL 1-201 population stimulated as in (A) is shown. Bar = 25 μm. (D) Calculated slopes of the C-tag TNF channel signal of cells stimulated as in (A) are depicted, while the mean value is highlighted in red. Representative data of three (A and C) independent experiments are shown. Statistics indicates significance by one-way ANOVA (D) with a Tukey correction for multiple testing: *** P<0.001, ns=not significant. Raw data are available in —source data 1.

Article Snippet: The following antibodies were purchased from commercial suppliers: Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific, Cat# A-11001, RRID:AB_2534069 and Cat# A-11008, RRID:AB_143165), anti-b-actin (C4) HRP (Santa Cruz Biotechnology, Cat# sc-47778 HRP, RRID:AB_271418), anti-Caspase-8 (1C12) (Cell Signaling Technology, Cat# 9746, RRID:AB_2275120), anti-ADAM10, CT (Millipore, Cat# AB19026, RRID:AB_2242320), anti-ADAM17 (D22H4) (Cell Signaling Technology, Cat# 6978, RRID:AB_1082838), anti-IκBα (L35A5) (Cell Signaling Technology, Cat# 4814, RRID:AB_390781), anti-phospho-IkBa (14D4) (Cell Signaling Technology, Cat# 2859, RRID:AB_561111), anti-TNF (D5G9) (Cell Signaling Technology, Cat# 6945, RRID:AB_10859375), CaptureSelect™ Alexa488 anti-C-tag conjugate (Thermo Fisher Scientific, Cat#7213252100), MLKL polyclonal antibody (Thermo Fisher Scientific, Cat# PA5-34733, RRID:AB_2552085), phospho-NF-κB p65 (Ser536) (93H1) rabbit mAb (Cell Signaling Technology, Cat# 3033, RRID:AB_331284), recombinant anti-MLKL (phospho S358) antibody (Abcam, Cat# ab187091, RRID:AB_2619685).

Techniques: Transfection