recombinant human adam 10  (R&D Systems)

Journal: Nature Communications

Article Title: Evolutionary regulation of human Fas ligand (CD95L) by plasmin in solid cancer immunotherapy

doi: 10.1038/s41467-025-60990-0

Figure Lengend Snippet: a Top: showing schematic and location of potential ADAM10 cleavage sites in huFasL. Bottom: RhFasL and huFasL were incubated with ADAM10 for indicated times followed by immunoblotting (reducing condition) with anti-FasL antibody. Representative blot is n = 1. b RhFasL and huFasL were incubated with plasmin (Pln) for indicated times followed by immunoblotting (reducing condition) for FasL and plasmin. Representative blot is n = 1, however it has been repeated multiple times in manuscript with additional parameters. c Schematic of genetic construction of his-tagged 1X and 2XFasL constructs described in ( d ). d Indicated RhFasL and huFasL were incubated with Pln for O/N followed by immunoblotting for FasL. Representative blot is n = 1. e The kinetic binding behavior of huFasL and RhFasL was analyzed against fixed and immobilized human plasmin at indicated pH values. f Schematic of myc-tagged membrane attached FasL ECDs. g Indicated membrane FasL-expressing cells were treated with Pln followed by immunoblotting against c-myc. Representative blot is n = 1. h Cell survival assay after HuFasL/RhFasL ± Pln treatment ( n = 3, three independent experimental repeats) Untreated vs. huFasL, **** p = <0.0001; Untreated vs. huFasL+Pln, * p = 0.0214; Untreated vs. RhFasL, **** p = <0.0001; Untreated vs. RhFasL+Pln, **** p = <0.0001 < 0.0001; Unpaired, two-sided parametric t-test with no adjustments. i Same as ( h ), except lysates were analyzed for caspase-8 and PARP. Representative blot is n = 1, however it has been repeated in vivo with additional parameters, see Figs. , . Error bars in ( h ) are presented as mean ± standard deviation (SD). In ( i ) u.c. indicates uncleaved, c. indicates cleaved.

Article Snippet: Recombinant HuFasL and RhFasL containing the extracellular FasL domain (aa 103–281) were incubated with recombinant human His-tagged ADAM10 (Catalog #: 936-AD, R&D Systems, USA) (1μ/20 μl reaction mixture) for indicated time at 37 °C in reaction buffer (25 mM Tris, 0.005% Brij-35; 2.5 μM ZnCl 2 , pH 8.8) as published earlier .

Techniques: Incubation, Western Blot, Construct, Binding Assay, Membrane, Expressing, Clonogenic Cell Survival Assay, In Vivo, Standard Deviation

Journal: ACS nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics.

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Figure 2. Inclusion of the Ly6G/C-specific VHH16 enhances the activation of the conditional antimicrobial therapeutic peptide. (A) Design of a conditional antimicrobial therapeutic for delivery of POL. (B) Characterization of the 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. The asterisk (*) denotes statistical significance (P < 0.05). Panel E was partly created with BioRender.com.

Article Snippet: Dye-labeled antimicrobial therapeutics (VHHα-(ABD)2-(EEG)6-Sx-POL-Cy7 or VHH16-ABD-(EEG)6S17-PNT4-sulfo-Cy7) were incubated with recombinant human ADAM10 (R&D Systems, MN, U.S.A.) at 10 μM and 250 nM final concentrations, respectively.

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

Journal: ACS nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics.

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Figure 3. Enhanced activation of a VHH16-targeted conditional antimicrobial therapeutic requires interaction with the Ly6G/C target as well as proteolytic activity of ADAM10. (A) Experimental timeline for in vivo evaluation of the 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 pretreatment with an excess either 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 density 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 a 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. The asterisk (*) denotes statistical significance (P < 0.05).

Article Snippet: Dye-labeled antimicrobial therapeutics (VHHα-(ABD)2-(EEG)6-Sx-POL-Cy7 or VHH16-ABD-(EEG)6S17-PNT4-sulfo-Cy7) were incubated with recombinant human ADAM10 (R&D Systems, MN, U.S.A.) at 10 μM and 250 nM final concentrations, respectively.

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

Journal: ACS nano

Article Title: Nanobody-Targeted Conditional Antimicrobial Therapeutics.

doi: 10.1021/acsnano.4c16007

Figure Lengend Snippet: Figure 5. Demonstration of VHH16-enhanced activation of a conditional antimicrobial therapeutic protein. (A) Design of a 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 a 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 the biodistribution and activation of VHH16-ABD-(EEG)6-S17-PNT4- sulfo-Cy7. Quantification of the total and activated fractions of 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 the therapeutic efficacy of VHH16-ABD-(EEG)6-S17-PNT4. (I) Quantification of bacterial burden from the treated lungs presented as log(cfu/g). The dotted line denotes the 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. The asterisk (*) denotes statistical significance (P < 0.05). The evaluation of efficacy was confirmed in two independent studies with similar results.

Article Snippet: Dye-labeled antimicrobial therapeutics (VHHα-(ABD)2-(EEG)6-Sx-POL-Cy7 or VHH16-ABD-(EEG)6S17-PNT4-sulfo-Cy7) were incubated with recombinant human ADAM10 (R&D Systems, MN, U.S.A.) at 10 μM and 250 nM final concentrations, respectively.

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

Journal: BMB Reports

Article Title: Recombinant ADAMTS1 promotes muscle cell differentiation and alleviates muscle atrophy by repressing NOTCH1

doi: 10.5483/BMBRep.2024-0109

Figure Lengend Snippet: Inhibitory effect of recombinant ADAMTS1 on ADAM10-mediated NOTCH1 activation. (A, B) ADAMTS1 knockdown C2C12 cells were treated with recombinant ADAMTS1 protein at a concentration of 100 ng/ml. Differentiated myotubes were visualized using Jenner’s staining (A), and the myotube length was measured (B). Scale bar: 500 μm. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Effect of ADAM10 on muscle cell differentiation. Recombinant ADAM10 protein was added at concentrations of 0.1, 1, 10, and 100 ng/ml. Differentiated myotube length was measured. ***P < 0.001. (D) Western blotting analysis of ADAM10-treated C2C12 cells. Quantification of protein expression using ImageJ software. ***P < 0.001. (E) Immunofluorescence staining of C2C12 cells. Cells were treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Scale bar: 100 μm. (F) Western blotting analysis of C2C12 cells treated with recombinant ADAM10 at 10 ng/ml and recombinant ADAMTS1 at 0 (untreated), 1, 10, 100, and 1000 ng/ml. Protein expression was quantified using ImageJ software. † P < 0.05, †† P < 0.01, ††† P < 0.001 compared to control group, *P < 0.05, **P < 0.01, ***P < 0.001 compared to recombinant ADAMTS1 untreated group. (G) Quantification of Hes1 mRNA expression. † P < 0.05 compared to control group, **P < 0.01 compared to the untreated group. Data are presented as the mean ± standard deviation. Statistical significance was tested using one-way ANOVA with Tukey’s post hoc test. Con, control; si-ctrl, control siRNA; si-ATS #1, ADAMTS1 siRNA #1; si-ATS #2, ADAMTS1 siRNA #2; rA, recombinant ADAMTS1.

Article Snippet: Recombinant ADAM10 was purchased from R&D systems (936-AD).

Techniques: Recombinant, Activation Assay, Knockdown, Concentration Assay, Staining, Cell Differentiation, Western Blot, Expressing, Software, Immunofluorescence, Control, Standard Deviation