Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: ADAM10 and ADAM17 are Robo4 sheddases. ( A ). Single-cell RNAseq transcriptome analysis of ADAM expression in dEC in adult C57BL/J mice . ADAM expression was normalized to GAPDH in the same cell, and the mean value in each mouse was calculated. The top 10 expressing ADAMs in dEC are plotted. The full list of analyzed ADAM s is included in the Methods. ( B ) Quantitative RT-PCR analysis determined the mRNA expressions of ADAM10 , ADAM17 , ADAMTS-4 , and ADAMTS-5 in a mouse dEC line, and the data were normalized to ADAM10 expression. ( C – F ) Pharmacological inhibition of ADAM10, ADAM17, or both blocked Robo4 shedding in dEC ( C ), mouse lung endothelial cells ( E ), and primary HUVECs ( F ) and led to corresponding increased cell surface Robo4 ( D ). The endothelial cells were treated with GI, TAPI-2, or GW at 6 μM or vehicle (DMSO) for 6 h, and sRobo4 in conditioned medium was assessed and normalized to full-length Robo4 in the cell lysate. The data was further normalized to the DMSO group for comparison. The dEC cell surface Robo4 was assessed by flow cytometry after staining with an anti-Robo4 ectodomain antibody. Anti-Robo4 IgG and naïve IgG staining are drawn in heavy-bright and thin-faint lines, respectively, with corresponding colors. ( G ) Knockdown (KD) of ADAM10 and ADAM17 . dECs were transiently transfected with scramble shRNA or shRNA against ADAM10 or ADAM17 , and ADAM10 and ADAM17 expression in the shRNA-treated cells were assessed by Western blot with corresponding specific antibodies. Black bars separate lanes that are nonadjacent in the same blot. ( H ) Knockdown of ADAM10 or ADAM17 each inhibited Robo4 shedding. sRobo4 in 6-h conditioned media was assessed by Western blot. The data represent 3 independent experiments and are presented as mean ± SD. The student’s t-test was performed for two-group comparisons. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Expressing, Quantitative RT-PCR, Inhibition, Flow Cytometry, Staining, Transfection, shRNA, Western Blot

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: Inhibition of ADAM10 and ADAM17 increases Robo4 C-terminal Fragment, and Robo4 co-localizes with ADAM10 and ADAM17 in endothelial cells. ( A ) Human Robo4-HA-FLAG expression. The expression of hRobo4-HA-FLAG in dEC lysate was probed with an anti-human Robo4 ectodomain antibody in Western blot. A 130 KDa band was detected. ( B ) Pharmacological inhibition of ADAM10 and ADAM17 decreased Robo4-CTF. hRobo4-HA-FLAG transiently expressing dECs were treated with vehicle control DMSO or GW (6 μM) with or without Mg132 (12 μM) or chloroquine diphosphate (CD, 50 μM). The cell lysates were probed with an anti-FLAG antibody. The hRobo4 CTF was normalized to β-actin and further normalized to the control group. GW and Mg132 decreased hRobo4-CTF. ( C ) Knockdown of ADAM10 or ADAM17 decreased Robo4 CTF. The dECs were co-transected with hRobo4-HA-FLAG and scrambled or ADAM knockdown constructs and then probed with an anti-FLAG antibody in western blot. ( D ) Inhibition of ADAM10 and ADAM17 decreased endogenous Robo4-CTF. Vehicle DMSO or GW (6 μM)-treated dECs were lysed and probed with an anti-mouse Robo4 CTF antibody in western blot. No Robo4 bands were detected in the Robo4 KO dEC cell lysate. ( E ) Robo4 co-localizes with ADAM10 and ADAM17. dECs were transiently expressed with hRobo4-HA-FLAG and stained for HA and endogenous ADAM10 or ADAM17 with corresponding antibodies. The merge yellow fluorescence shows Robo4-HA (green) co-localized with ADAM10 or ADAM17 (red). Robo4-HA-FLAG co-localized with ADAM10 or ADAM17 with overlap coefficients of 0.900 and 0.834, respectively. The data represent 3 independent experiments and are presented as mean ± SD. The student's t-test was performed for a two-group comparison. *p < 0.05; **p < 0.01.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Inhibition, Expressing, Western Blot, Construct, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Robo4 is constitutively shed by ADAMs from endothelial cells and the shed Robo4 functions to inhibit Slit3-induced angiogenesis

doi: 10.1038/s41598-022-08227-8

Figure Lengend Snippet: sRobo4 generation and its role in angiogenic Slit3-Robo4 signaling. Under the unstimulated condition, the Robo4 ectodomain is constitutively cleaved by ADAM10 and ADAM17 to generate sRobo4. The generated sRobo4 blocks Slit3-Robo4 interaction, thereby inhibiting angiogenic Slit3 signaling. Meanwhile, Slit3 inhibits Robo4 shedding by inducing Robo4 internalization to shield the receptor from shedding.

Article Snippet: Anti-N-terminal mouse Robo4 antibody (Abcam, ab10547), anti-N-terminal human Robo4 antibody (R&D Systems, MAB2454), anti-FLAG antibody (Thermo Fisher Scientific, 14-6681-82), anti-HA antibody (Chromotek, #7c9-100), anti-intracellular Robo4 domain antibody (Santa Cruz Biotechnology, sc46497), anti-ADAM10-pro antibody (Abcam, ab39178), anti-ADAM10 antibody (Bioss, bs-3574R; LSbio, C497146-200; Novus, #NBP1-76973), anti-ADAM17 antibody (Bioss, 4236R; Novus #NBP2-61719), anti-Synecan-1 antibody (Santa Cruz Biotechnology, sc-5632), anti-actin antibody (Sigma Aldrich, A2228), anti-GAPDH antibody (R&D Systems, AF5718), polyclonal anti-Unc5B antibody (R&D Systems, AF1006), anti-N-terminal CDH5 (Thermo Fisher Scientific, 14-1441-82), anti-CD31 (BD, #550274), HRP-conjugated anti-His antibody (Alpha Diagnostic International, HISP12-HRP), HRP-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, sc-2030), HRP-conjugated donkey anti-goat IgG antibody (Santa Cruz Biotechnology, sc-2020), and HRP-conjugated goat anti-mouse IgG antibody (Invitrogen, 62-6520).

Techniques: Generated

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were either treated with 0.1 μg/mL PMA or 5 μM ionomycin (Iono) for 30 min, or infected with CLJ1 (90 min), or left untreated/uninfected (NI). Cellular extracts were analysed by Western blot using E-cadherin and β-actin antibodies. FL, full-length; CTF, C-terminal fragment. The experiment was performed twice. B . A549 cells (left) or HUVECs (right) were pre-treated with DMSO, the general metalloprotease inhibitor GM6001 (10 μg/mL) or the specific ADAM10 inhibitor GI254023X (5 μM) and then incubated with CLJ1 or IHMA87 (90 min), or uninfected (NI). Cellular extracts were analysed as above. The experiment was performed twice for A549 and 3 times for HUVECs. C . A549 or ADAM10-deficient A549 (A549 ADAM10 -/- ) cells were incubated with either CLJ1 or IHMA87. Cellular extracts were prepared at different time points post-infection as indicated and analysed by Western blot (left). The right panel shows the FACS analysis of ADAM10 surface expression of both cell lines, as well as the negative control. The experiment was performed 3 times. D . Similar experiment with HUVECs, either transfected with ADAM10 siRNA or untreated. The experiment was performed twice.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Infection, Western Blot, Incubation, Expressing, Negative Control, Transfection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: Plasma membrane rupture was monitored by LDH release in the supernatant. A549 or A549 ADAM10 -/- cells were incubated for 5 hours with IHMA87, IHMA87Δ exlA or IHMA87Δ exlA/exlA strains. The supernatants were the tested for LDH activity. The histograms show the mean ± s.d. of triplicates. The data are representative of 3 experiments.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Incubation, Activity Assay

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells were incubated with various concentrations of TFP, as indicated, to impede calmodulin interaction with ADAM10. Ionomycin was used as positive controls. E-cadherin cleavage was assessed by Western blot. The experiment was performed twice. B . Western blot analysis of A549 E-cadherin contents after infection with CLJ1 or IHMA87, in presence or absence of BAPTA-AM. Both experiments were performed 3 times. C . LDH release of A549 cells infected with either CLJ1 or IHMA87, in presence/ absence of BAPTA-AM. Student’s t-test showed significance between the two treatments for both CLJ1 and IHMA87 data (p-values indicated above the bars). The experiment was performed 3 times.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Western Blot, Infection

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: A . A549 cells (left) or HUVECs (right) were incubated with the S . marcescens ShlA-secreting strain Db11, or with the non-ShlA-secreting mutant 21C4. Cellular extracts were analysed for their E- or VE-cadherin contents. The experiment was performed twice for the left panel and once for the right panel. B . Similar analysis using A549 ADAM10 -/- . The experiment was performed once. C . Similar analysis using A549 cells, in presence/ absence of BAPTA-AM. D-G . Intracellular Ca 2+ contents and plasma membrane permeability were measured using Fluo3-AM and Draq7 fluorescent probes, respectively. A549 cells ( D,F ) and HUVECs ( E,G ) were infected with Db11 ( D,E ) or 21C4 ( F,G ) and fluorescence was recorded on both channels by videomicroscopy. Five cells were analysed in each case; the Fluo3 intensities are represented by straight lines and the Draq7 intensities by dashed lines, using the same colour code for one cell. Data are representative of 8 and 5 independent experiments for A549 and HUVECs, respectively.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Incubation, Mutagenesis, Clinical Proteomics, Membrane, Permeability, Infection, Fluorescence

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation

doi: 10.1371/journal.ppat.1006579

Figure Lengend Snippet: In uninfected cells, pro-ADAM10 is associated with calmodulin, preventing its maturation and export to the plasma membrane. Pore formation by ExlA or ShlA induces a massive Ca 2+ influx in host cells. Intracellular Ca 2+ interacts with the Ca 2+ -binding protein calmodulin, which detaches from pro-ADAM10, allowing its maturation to m-ADAM10. m-ADAM10 cleaves E- and VE-cadherin in epithelial and endothelial cells, respectively, provoking intercellular junction rupture.

Article Snippet: Three days after transfection, cells were analysed by FACScalibur flow cytometer (Becton Dickinson) after staining with ADAM10 antibody (R&D Systems) and anti-mouse-Alexa 488 antibody (Molecular Probes).

Techniques: Clinical Proteomics, Membrane, Binding 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:

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: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 2 | Influence of metalloproteinases on the expression and release of CXCL16. (a) Analysis of soluble CXCL16 released from primary TALDCs with a CXCL16-specific ELISA. Cells were preincubated with different proteinase inhibitors for 15 min before IFN-g (24 ng/ml) was added for 24 h. Application of IFN-g (24 ng/ml) increased the amount of soluble CXCL16 in the supernatants of TALDCs. The ADAM10-specific metalloproteinase inhibitor, GI254023X (GI), and the broad-spectrum metalloproteinase inhibitors, GM6001 and TAPI-2, reduced the IFN-g-induced CXCL16 release. Data are mean±s.d. (n ¼ 3); ***Po0.001 versus control; ###Po0.001, ##Po0.01; #Po0.05 versus cytokine-treated cells. (b) Inhibition of metalloproteinases increased cellular CXCL16 protein expression in TALDCs. Fifteen minutes before application of IFN-g, cells were pretreated with the indicated concentration of the metalloproteinase inhibitors GI254023X, GM6001, and TAPI-2. CXCL16 protein levels were measured by western blot analysis and b-actin was used as a loading control. (c) Knockdown of CXCL16 with CXCL16-specific siRNA in TALDCs is shown by western blot analysis. (d) Application of pharmacological inhibitors of metalloproteinases increased the amount of cellular CXCL16. Cell lysates were isolated as described under Materials and Methods, and 15 mg total proteins were used to measure the amount of cell-expressed CXCL16 by CXCL16-specific ELISA. Data are mean þ s.d. (n ¼ 5). Statistically significant release of CXCL16 (Po0.001) is indicated by asterisk. (e) Suppression of ADAM10 protein levels by RNA interference in the presence and absence of IFN-g. Cell lysates of TALDCs were prepared 48 h after transfection with siRNA specific for ADAM10 and an unspecific siRNA. Mock-transfected cells treated with the transfection lipid in the absence of siRNA were used as a control. Western blot analysis of ADAM10 protein expression was performed. Blots were reprobed with an antibody specific for b-actin as a loading control. (f) Inhibition of ADAM10 by siRNA reduced the IFN-g-stimulated CXCL16 release in TALDCs. Supernatants of siRNA-treated and IFN-g- stimulated or unstimulated cells were analyzed for soluble CXCL16 using a CXCL16-specific ELISA (n ¼ 3); ***Po0.001 versus control; ###Po0.001 versus IFNg-treated cells.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Inhibition, Concentration Assay, Western Blot, Knockdown, Isolation, Transfection

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 3 | Localization of ADAM10 protein in normal human kidney. (a) In the overview, ADAM10 protein is mainly expressed in tubular cells, but expression was also found in glomerular cells. Serial sections of normal renal tissue represented constitutive ADAM10 expression in aquaporin-2 (e), calbindin D-28K (f), and Tamm–Horsfall glycoprotein (g) expressing tubular profiles, which correspond to CD, (DCT, CNT), and TAL, respectively. Proximal tubules were devoid of any specific ADAM10 staining. (h) Immunofluorescent detection of ADAM10 (red fluorescence) in aquaporin-2-positive principal cells of the CD (green fluorescence). A prominent overlap of both signals in the same cells can be seen, indicated in the examples by asterisks. In contrast, arrows indicate cells negative for either aquaporin-2 or ADAM10, thus representing presumable intercalated cells. (i) ADAM10 is not expressed in the intercalated cells of the CD. Note: arrows represent H þ-ATPase-expressing intercalated cells (green colour) that do not express ADAM10 (red colour); compare with the merged view. (j) Confocal immunofluorescence analysis of ADAM10 and CXCL16 expression in a cortical CD. Tissue section was stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. Both signals can be confined to same segment-specific cells; see merged picture on the right.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Staining, Fluorescence, Immunofluorescence

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 4 | Supernatants from ADAM10- and CXCL16 siRNA-transfected TALDCs decreased the chemotaxis of Jurkat T cells. (a) Surface expression of the chemokine receptor CXCR6 in Jurkat T cells analyzed by fluorescence-activated cell sorting analysis. (b) Inhibition of CXCL16 protein expression by siRNA fully abolished release of CXCL16 in TALDCs. Cells were transfected with different siRNA duplexes for inhibition of ADAM10 or CXCL16, an unspecific siRNA (scrambled), or not transfected with siRNA (mock). A total of 24 h after transfection, TALDCs were stimulated with IFN-g for 24 h, where indicated, and supernatants were collected. Soluble CXCL16 was determined by a CXCL16-specific ELISA (n ¼ 4). Data are mean±s.d.; ***Po0.001 versus control; ###Po0.001 versus IFN-g-treated cells. (c) Decrease of soluble CXCL16 correlates with a reduction in the migration of Jurkat T cells. Supernatants shown in Figure 5b were used for chemotaxis assays (n ¼ 2). Recombinant CXCL16 induced migration of CXCR6-expressing Jurkat T cells about threefold; **Po0.01; *Po0.05 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Transfection, Chemotaxis Assay, Expressing, Fluorescence, FACS, Inhibition, Enzyme-linked Immunosorbent Assay, Control, Migration, Recombinant

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 5 | Increased urinary CXCL16 correlated with focally apical CXCL16 expression in tubular cells of renal allografts with the histopathological diagnosis of ATN. Six allograft biopsies of patients diagnosed with ATN (cases A1–A6; clinical parameters and CXCL16 analysis are shown in Table 1) were analyzed by CXCL16 immunohistochemical analysis (a–f) and with ADAM10 (green colour) and CXCL16 (red colour) double immunofluorescence staining (g–l). Notably, all patients showed focally increased apical CXCL16 expression in renal tubuli. Strongest apical CXCL16, seen in patient A4 (d), correlated with the highest amount of urinary CXCL16 (Table 1). In contrast, ADAM10 expression (green colour) was not significantly changed in ATN patients (A1–A6) compared with normal kidney (g–l). (m) Urinary CXCL16 measured by a CXCL16-specific ELISA in healthy volunteers (normal), in patients with IR, and in patients with ATN. Data are mean±s.d.; **Po0.01 versus control.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Double Immunofluorescence Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Kidney international

Article Title: Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

doi: 10.1038/ki.2008.181

Figure Lengend Snippet: Figure 6 | Increased ADAM10 expression in allograft biopsies of kidney transplant patient with the clinical and histopathological diagnosis of acute IR. Renal allograft biopsies of patients diagnosed with IR were analyzed by double immunofluorescence for ADAM10 and CXCL16 expression in comparison with normal kidney (upper panel). Clinical features and ADAM10 analysis of patients are listed in Table 2. Tissue sections were stained with Alexa Fluor 488 and Cy3 secondary antibodies to visualize the localization of CXCL16 (red) and ADAM10 (green) proteins, respectively. In some tubuli of patient B1 (marked with arrows), strong basolateral ADAM10 expression and no CXCL16 expression could be seen (middle panel). In contrast, in few tubuli of the same patient (marked with a star) both molecules colocalized. In the lower panel (patient B5), strong coexpression of ADAM10 and CXCL16 in a tubule is visible, accompanied with interstitial inflammatory infiltrates.

Article Snippet: Recombinant human CXCL16, recombinant human IFN-g, anti-human ADAM10 ectodomain antibody, and anti-human CXCR6 antibody were obtained from R&D Systems (Wiesbaden, Germany).

Techniques: Expressing, Biomarker Discovery, Immunofluorescence, Comparison, Staining