anti human adam10 ecd mouse mab igg2b  (R&D Systems)

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Biochemical evidence for ADAM10-mediated CA IX ECD cleavage. (A) Verification of the cleavage activity of rhADAM10 catalytic domain towards the fluorogenic peptide Mca-K-P-L-G-L-Dpa-A-R-NH 2 . The peptide was used at the final concentration of 10 µM in a total of 100 µl reaction mixture with 10 ng of the rhADAM10. The cleavage was allowed to proceed for 30, 40, 50 and 120 min. Time-related increase of the fluorescence emitted from the peptide proves that rhADAM10 was active in comparison to negative control without rhADAM10. (B) ELISA analysis of CA IX ECD in culture medium obtained from CHOwt-FL-CA IX and CHOwt-NS-CA IX cells transiently expressing FL-CA IX and NS-CA IX, respectively. Transfected cells were treated with rhADAM10 (500 ng/ml) for 24 h (Data were analyzed by Student's t-test). (C) Incubation of CHOwt-FL-CA IX cells with ADAM17 activator PMA (20 µM, 3 h), ADAM10 activator IONO (1 µg/ml, 3 h) or rhADAM10 (500 ng/ml, 24 h). Data were analyzed using one-way ANOVA followed by Dunnett's test. Experiments were performed in triplicates and repeated twice. The results were expressed as the mean ± SD. **P<0.01 and ***P<0.001. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; rhADAM10, recombinant human ADAM10; ECD, ectodomain; FL, full-length; NS, non-shed; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; ns, non-significant.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activity Assay, Concentration Assay, Fluorescence, Comparison, Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Incubation, Recombinant

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Co-localization of ADAM10 and CA IX in C33a-FL-CA IX cells. Immunofluorescence of (A) C33a-FL-CA IX cells expressing CA IX, and (B) control C33a-neo cells using ADAM10-specific antibody. Nuclei were counterstained with DAPI. PLA performed in (C) C33a-FL-CA IX and (D) C33a-neo cells using rabbit anti-human CA IX-specific antibody and mouse anti-human ADAM10 antibody. Red PLA signal indicating the interaction of CA IX with ADAM10 was clearly visible only in C33a cells expressing FL CA IX. Experiment was performed in triplicates and repeated twice. Magnification, ×630. Scale bar, 10 µm. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX; PLA, proximity ligation assay; FL, full-length.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Immunofluorescence, Expressing, Control, Proximity Ligation Assay

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Localization of ADAM10 (green) in response to CA9hu-1 antibody-induced CA IX internalization (red). C33a-FL-CA IX cells were pre-incubated either with anti-ADAM10 antibody alone (ctrl, A-D) or with anti-ADAM10 antibody together with the internalization-inducing anti-CA IX antibody CA9hu-1 (E-H) 30 min at 4°C. Plasma membrane staining signal for ADAM10 and CA IX was observed at all time points in the absence of CA9hu-1 pre-treatment. On the other hand, CA9hu-1-induced internalization of CA IX as well as ADAM10 was visible after 2 and 4 h at 37°C (E and F), while both molecules showed recycling to plasma membrane after 8 and 24 h. Overlapped staining signals were evident in all samples. Magnification, ×630. Scale bar, 20 µm. Experiment was performed in triplicates and repeated twice. ADAM, a disintegrin and metalloproteinase; CA IX, carbonic anhydrase IX.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Incubation, Clinical Proteomics, Membrane, Staining

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: GI-induced internalization of ADAM10 and targeting of ADAM10 by RNA interference or by a dominant-negative ADAM10 mutant ∆MP. (A-H) C33a-FL-CA IX cells were pre-incubated with anti-ADAM10 antibody at 4°C. (A-D) Cells showed the plasma membrane staining signal for ADAM10 (green) in absence of GI at 37°C. (E-H) GI-induced internalization of ADAM10 was clearly visible as cytoplasmic staining signal in all incubation periods at 37°C. (I) Direct targeting of ADAM10 was performed by RNA interference and (J) expression of a dominant-negative mutant ∆MP with and without GI treatment. Control cells were transfected with either an esiRNA targeting RLUC or an empty vector (pcDNA3.1). Culture media collected from transfected cells incubated in presence and absence of GI for 24 h were collected and subjected to ELISA analysis for detection of CA IX ECD. Experiment was performed in triplicates and repeated six times. Data were analyzed by one-way ANOVA followed by Dunnett's test. Results were expressed as the mean percentage of CA IX ECD shedding with control cells set as 100% ± SD. ***P<0.001. GI, GI254023X; ADAM, a disintegrin and metalloproteinase; ∆MP, dominant-negative mutant of ADAM10; esiRNA, enzymatically-prepared small interfering RNA; CA IX, carbonic anhydrase IX; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Dominant Negative Mutation, Mutagenesis, Incubation, Clinical Proteomics, Membrane, Staining, Expressing, Control, Transfection, esiRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Small Interfering RNA

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Additive effect of ADAM10 and ADAM17 activation/inhibition. (A) Quantitative PCR analysis of ADAM10 and ADAM17 mRNA levels in C33a-FL-CA IX and C33a-NS-CA IX cells normalized to the level of β-actin mRNA. (B) Western blot analysis of ADAM10, ADAM17 and CA IX protein in C33a-FL-CA IX and C33a-NS-CA IX cells. The anti-actin antibody was used as a loading control. (C and D) ELISA analysis of CA IX ECD in culture medium collected from C33-FL-CA IX cells after the treatment with IONO (1 µg/ml), PMA (20 µM), IONO + PMA (1 µg/ml; 20 µM), D1(A12) antibody (200 nM), GI inhibitor (1 µM) or D1(A12) + GI (200 nM; 1 µM) for 3 h in comparison to non-treated cells (CTRL). Experiment was performed in triplicates and repeated two times. Data were analyzed by (A) Student's t-test and (C and D) one-way ANOVA followed by Dunnett's test. Results were expressed as the mean relative levels of mRNA or CA IX ECD ± SD. ***P<0.001. ADAM, a disintegrin and metalloproteinase; FL, full-length; CA IX, carbonic anhydrase IX; NS, non-shed; ECT, ectodomain; IONO, ionomycin; PMA, phorbol 12-myristate 13-acetate; GI, GI254023X.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Activation Assay, Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Oncology Reports

Article Title: ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non-redundantly to ADAM17

doi: 10.3892/or.2022.8464

Figure Lengend Snippet: Scheme illustrating CA IX ECD shedding by ADAM10 and ADAM17 proteinases. Picture depicts differences in regulatory components that differentially affect ADAMs biosynthesis and processing at various levels of their expression and activation and thereby can potentially influence CA IX ECD cleavage. Based on ( , – ). CA IX, carbonic anhydrase IX; ADAM, a disintegrin and metalloproteinase; ECD, ectodomain.

Article Snippet: For detection of ADAM10, cells cultured on glass coverslips were incubated with anti-human ADAM10 ECD mouse Mab IgG2B Clone 163003 (R&D Systems, Inc.; cat. no. MAB1427, 1:100 in cultivation medium) for 1 h at 37°C, gently washed with PBS and fixed with methanol for 5 min at −20°C.

Techniques: Expressing, Activation Assay

Journal: eLife

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

doi: 10.7554/eLife.99172

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

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

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

Journal: eLife

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

doi: 10.7554/eLife.99172

Figure Lengend Snippet:

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

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

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: GPVI cleavage is dependent on Tspan15 and Tspan33 in transfected HEK-293T cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+), or empty vector (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest. Cells were then lysed in 1% Triton X-100 and lysates subjected to Western blotting with an anti-Myc antibody. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective WT controls in each stimulation condition (***, p < 0.001; ****, p < 0.0001). ( B ) ADAM10 surface expression on the cell types described in panel A were analyzed by flow cytometry. Geometric mean fluorescence intensity was used to assess surface ADAM10 levels and data were made relative to WT values. Error bars represent the standard error of the mean from three independent experiments. Average geometric mean intensities were arcsine transformed and then statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to WT (****, p < 0.0001). The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Transfection, Knock-Out, Double Knockout, Expressing, Construct, Plasmid Preparation, Western Blot, Transformation Assay, Comparison, Flow Cytometry, Fluorescence, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15 and Tspan33 are required for cleavage of endogenous GPVI in HEL cells. ( Ai ) Wild-type (WT), ADAM10-knockout (A10 KO), Tspan14-knockout (T14 KO), Tspan15-knockout (T15 KO), Tspan33-knockout (T33 KO) and GPVI-knockout (GPVI KO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were stimulated with 2 mM NEM (+) for 1 h before harvest. Cells were lysed in 1% Triton X-100 and separated by SDS-PAGE. Lysates were probed with a mouse anti-human GPVI antibody (11A7), which recognises the extracellular region of GPVI and therefore only the full-length protein (top panel) and a rabbit anti-human GPVI antibody, which recognises the cytoplasmic tail of GPVI (bottom panel). ( Aii ) Relative GPVI cleavage was calculated by normalising the cleaved fragment signal to the full-length GPVI; the NEM-treated WT was arbitrarily set at 100. Error bars represent the standard error of the mean from three independent experiments. Data were normalised by arcsine transformation and statistically analyzed using a two-way ANOVA followed by a Bonferroni multiple comparison test, compared to WT (***, p < 0.001; ****, p < 0.0001). ( Bi ) WT, T33 KO and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with PMA and subjected to lysis and Western blotting as described in panel ( Ai ). ( Bii ) Relative GPVI cleaved was quantitated and statistically analyzed as described in panel ( Aii ). ( C ) ADAM10 surface expression on PMA-differentiated cells used in panels A and B were analyzed by flow cytometry and quantitated as described in B. The data shown are from single CRISPR/Cas9 knockout clones, but similar data were generated using a second set of clones generated using different guide RNA sequences (data not shown).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Knock-Out, SDS Page, Transformation Assay, Comparison, Double Knockout, Lysis, Western Blot, Expressing, Flow Cytometry, CRISPR, Clone Assay, Generated

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Tspan15/ADAM10 is the most efficient scissor for GPVI in HEK-293T cells. ( Ai ) Tspan15/33 double knockout (T15/33 dKO) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged human GPVI and FcRγ (+) or empty vector (–), and FLAG-tagged human Tspan14, Tspan15, Tspan33 or empty vector control (–). Cells were treated with 2 mM NEM (+), or vehicle control (–) for 30 min prior to harvest, and then lysed in 1% Triton X-100 followed by anti-Myc and anti-FLAG Western blotting. ( Aii ) The percentage of cleaved GPVI from Ai was calculated. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a two-way ANOVA followed by a Dunnett’s multiple comparison test, compared to their respective non-transfected controls in each stimulation condition (*, p < 0.05, **, p < 0.01; ****, p < 0.0001).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Double Knockout, Transfection, Expressing, Construct, Plasmid Preparation, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The extracellular region of Tspan15 is required for efficient GPVI cleavage and the C-terminus is inhibitory. ( A ) Schematic of N-terminally FLAG-tagged Tspan14 (grey) and Tspan15 (black) chimeras, where the entire extracellular region (EC), cytoplasmic domain (Cyto) or C-terminal tail (C) were exchanged. Truncation of both the N- and C-terminal tails is indicated by ΔNC. Ovals represent N-glycosylation sites. ( B ) Wild-type (WT) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI and FcRγ (+) or empty vector (–). In addition to GPVI and FcRγ, Tspan14/15/33 triple knockout (T14/15/33 tKO) HEK-293T cells were co-transfected with HA-tagged ADAM10 alone, or in combination with FLAG-tagged Tspan14 and Tspan15 constructs described in panel A. Cells were lysed in 1% Triton X-100 followed by Western blotting with anti-Myc, anti-FLAG and anti-HA antibodies (top panels). The percentage of cleaved GPVI was calculated (lower panel). Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to T14/15/33 tKO cells transfected with wild-type Tspan15 and ADAM10 (***, p < 0.001; ****, p < 0.0001).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Triple Knockout, Western Blot, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: The capacity of Tspan15 wild-type and mutant forms to promote GPVI cleavage is unrelated to the extent to which they co-localise with GPVI. Tspan14/15/33 triple knockout HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI, FcRγ, ADAM10 and FLAG-tagged Tspan14 or 15 chimeras that are described in detail in A. Cells were fixed and stained with anti-Myc (GPVI–Myc, magenta) and anti-FLAG (FLAG-Tspan, green) antibodies. ( Ai ) The middle planes of the cells were imaged using Airyscan confocal microscopy in super-resolution mode (scale bar 5 μm). No signal was detected in either channel in the empty vector-transfected cells (data not shown). ( Aii ) The degree of co-localization between GPVI–Myc and FLAG-TspanC8s is presented as the percentage of co-localizing pixels in the GPVI–Myc (magenta) channel. Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation before being statistically analyzed by a one-way ANOVA, followed by Dunnett’s multiple comparison tests, compared to cells transfected with wild-type Tspan15 (**, p < 0.01; ****, p < 0.0001). ( B ) Scatter plot summarising the relationship between degree of co-localization, expressed as the average of data presented in panel Aii , and percentage of GPVI cleaved, expressed as mean of the quantitation presented in B.

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Mutagenesis, Triple Knockout, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Plasmid Preparation, Transformation Assay, Comparison, Quantitation Assay

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Co-localization between ADAM10 and GPVI is similar in Tspan14-knockout and Tspan15/33 double knockout HEL cells. Wild-type (WT), Tspan14-knockout (T14 KO) and Tspan15/33 double knockout (T15/33 dKO) HEL cells were treated with 6.2 ng/mL PMA for 72 h. Cells were fixed and stained with anti-ADAM10 mAb (11G2, magenta) and an antibody against the extracellular region of GPVI (336A9, green). Images of the ( A ) basal membrane and ( B ) middle section of the cells were captured using Airyscan confocal microscopy in super-resolution mode (top panels; scale bar 5 μm). No ADAM10 signal was detected in control ADAM10-knockout cells and no GPVI signal was detected in control GPVI-knockout cells (data not shown). The degree of co-localization between ADAM10 and GPVI is presented as the percentage of co-localizing pixels in the ADAM10 (magenta) channel (lower panels). Data are representative of 15 fields of view from three independent experiments and are normalized by arcsine transformation and statistically analyzed by a one-way ANOVA, followed by Bonferroni’s multiple comparison tests (n.s., not significant; *, p < 0.05).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Knock-Out, Double Knockout, Staining, Membrane, Confocal Microscopy, Transformation Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Platelet Collagen Receptor GPVI Is Cleaved by Tspan15/ADAM10 and Tspan33/ADAM10 Molecular Scissors

doi: 10.3390/ijms23052440

Figure Lengend Snippet: Evidence that cut site position on GPVI contributes to specific cleavage by Tspan15/ADAM10 and Tspan33/ADAM10. ( A ) Amino acid alignment of wild-type GPVI and the stalk extension mutants, GPVI+5 and GPVI+10: ADAM10 cut site is denoted by ^; residues inserted are highlighted in grey; transmembrane region is underlined. ( B ) Wild-type (WT) HEK-293T cells were transfected with expression constructs for C-terminally Myc-tagged GPVI and FcRγ (+) or empty vector (–). ADAM10/17 double knockout (A10/17 dKO) HEK-293T cells were co-transfected with GPVI and FcRγ, HA-tagged ADAM10 alone, or in combination with FLAG-tagged TspanC8s (Tspan5, 10, 14, 15, 17 or 33). Cells were lysed in 1% Triton X-100 followed by Western blotting with anti-Myc, anti-FLAG and anti-HA antibodies (data not shown). The percentage of cleaved GPVI was calculated. Cleavage assays for ( C ) GPVI+5 and ( D ) GPVI+10 was performed as described in panel B. Error bars represent the standard error of the mean from three independent experiments. Data were arcsine-transformed and statistically analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparison test, compared to A10/17 dKO cells transfected with ADAM10 alone (*, p < 0.05; **, p < 0.01, ***, p < 0.001; ****, p < 0.0001).

Article Snippet: For flow cytometry of ADAM10 on HEK-293T, cells were stained with 10 μg/mL allophycocyanin (APC)-conjugated mouse anti-human ADAM10, or the equivalent negative control mouse IgG (R&D Systems, Abingdon, UK).

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Double Knockout, Western Blot, Transformation Assay, Comparison

Journal: eLife

Article Title: Microbiota-derived short chain fatty acids modulate microglia and promote Aβ plaque deposition

doi: 10.7554/elife.59826

Figure Lengend Snippet: Figure 3. Short chain fatty acids (SCFA) mildly increase amyloidogenic processing. (A) Western blot analysis and (B) its densitometry quantification of 3 months old brain homogenates of control (Ctrl)- and SCFA-supplemented germ-free (GF) APPPS1 animals. The Ab level is significantly increased in SCFA group in comparison to Ctrl, despite unaffected APP FL levels. APP CTFs show a decreased C83 (**) to C99 (*) ratio. We could not detect alterations in protein levels of secretases involved in APP processing (ADAM10, BACE1, and g-secretase/PSEN1). m = ADAM10 mature form; im = ADAM10 immature form. Data represent mean ± SEM (unpaired T-test; n(Ctrl) = 3, n(SCFA) = 3). (C) Upper panel: g-Secretase reconstituted into lipid vesicles was incubated at 37˚C together with the C99-based substrate C100-His6 in the presence of increasing doses of a SCFA mixture (375 and 750 mM final concentration of total SCFA of an equimolar mixture of Na-acetate, Na-propionate, and Na-butyrate) for 24 hr. Production of Ab was analyzed by immunoblotting. g-Secretase inhibitor (GSI) L-685,458 (0.4 mM) was used as a negative control. No alterations in Ab levels were detected in the presence of SCFA. Lower panel: Qualitative analysis of individual Ab species via Tris-Bicine-Urea SDS-PAGE reveals that SCFA treatment does not alter the ratio among the different Ab species (Ab37-38-40-42/43) suggesting no direct effects on modulation of g-secretase cleavage. (D) Aggregation kinetics of monomeric Ab40 recorded by the increase in fluorescence of Thioflavin T incubated with either 30 mM NaCl (Ctrl) or 30 mM SCFA mixture do not show any significant difference, suggesting that SCFA do not directly modify Ab fibrillarization. Data points represent mean ± SD from three independent experiments.

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus musculus) APPPS1 doi: 10.1038/sj.embor.7400784 MGI:3765351 C57BL/6 background Antibody Anti-IBA1 (guinea pig polyclonal) SYSY Cat# 234 004, RRID:AB_2493179 IHC (1:500) Antibody Anti-IBA1 (rabbit polyclonal) Wako Cat#:019–19741, RRID:AB_839504 IHC (1:500) Antibody Anti-CD68 (rat monoclonal) Bio-Rad Cat#:MCA1957G, RRID:AB_324217 IHC (1:500), WB (1:1000) Antibody Anti-Amyloid Y188 (rabbit monoclonal) Abcam Cat#:1565–1, RRID:AB_562042 WB (1:1000) Antibody Anti-Amyloid beta 3552 (rabbit polyclonal) doi: 10.1523/JNEUROSCI.5354–05.2006 IHC (1:500), WB (1:2000) Antibody anti-beta-Amyloid, 6E10 (mouse monoclonal) BioLegend Cat# 803002, RRID:AB_2564654 IHC (1:500) Antibody Anti-Presenilin 1 (NT1) (mouse monoclonal) BioLegend Cat# SIG-39194–500, RRID:AB_10720504 WB (1:1000) Antibody Anti-Human Adam10 (mouse monoclonal) R and D Systems Cat# MAB1427, RRID:AB_2223057 WB (1:1000) Antibody Anti-BACE1 (rabbit monoclonal) Epitomics Cat# 2882–1, RRID:AB_2061494 WB (1:1000) Antibody Anti-Calnexin (rabbit monoclonal) Stressgen Cat# ADI-SPA-860, RRID:AB_10616095 WB (1:1000) Continued on next page Colombo et al. eLife 2021;10:e59826.

Techniques: Western Blot, Control, Comparison, Incubation, Concentration Assay, Negative Control, SDS Page, Fluorescence