goat anti ephrin a4  (R&D Systems)

Journal: Medicine

Article Title: Association of microRNA-155, interleukin 17A, and proteinuria in preeclampsia

doi: 10.1097/MD.0000000000006509

Figure Lengend Snippet: (A–C) Urine nephrin expression at different urine protein levels.

Article Snippet: After washing with PBS, sections were blocked with 1% bovine serum albumin for 30 minutes and stained with goat anti-human nephrin (1:50; R&D Systems, Minneapolis, MN) for 12 hours at 4°C, followed by rhodamine-conjugated rabbit anti-goat immunoglobulin G (1:100; Santa Clara, CA).

Techniques: Expressing

Journal: Medicine

Article Title: Association of microRNA-155, interleukin 17A, and proteinuria in preeclampsia

doi: 10.1097/MD.0000000000006509

Figure Lengend Snippet: Nephrin and podocyte apoptosis expression in different transfected groups.

Article Snippet: After washing with PBS, sections were blocked with 1% bovine serum albumin for 30 minutes and stained with goat anti-human nephrin (1:50; R&D Systems, Minneapolis, MN) for 12 hours at 4°C, followed by rhodamine-conjugated rabbit anti-goat immunoglobulin G (1:100; Santa Clara, CA).

Techniques: Expressing, Transfection

Journal: Medicine

Article Title: Association of microRNA-155, interleukin 17A, and proteinuria in preeclampsia

doi: 10.1097/MD.0000000000006509

Figure Lengend Snippet: (A–C) Urine nephrin expression at different urine protein levels.

Article Snippet: Sections were incubated with a primary polyclonal goat anti-human nephrin antibody against the intracellular domain of nephrin (1:50; R&D Systems), then with a secondary gold-conjugated (10 nm) rabbit anti-goat secondary antibody (1:100; DAKO, DK).

Techniques: Expressing

Journal: Medicine

Article Title: Association of microRNA-155, interleukin 17A, and proteinuria in preeclampsia

doi: 10.1097/MD.0000000000006509

Figure Lengend Snippet: Nephrin and podocyte apoptosis expression in different transfected groups.

Article Snippet: Sections were incubated with a primary polyclonal goat anti-human nephrin antibody against the intracellular domain of nephrin (1:50; R&D Systems), then with a secondary gold-conjugated (10 nm) rabbit anti-goat secondary antibody (1:100; DAKO, DK).

Techniques: Expressing, Transfection

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: Clinical and molecular details of patients

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques:

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: A. Expression of ephrinA4 was measured on the surface of CLL cells from 30 samples by flow cytometry analysis (MFI, mean fluorescence intensity) and (i) compared between samples according to disease parameters and cytogenetic characteristics or (ii) correlated with survival outcome of TM cells in LApos (left) and LAneg (right) cases. R: Spearman's correlation coefficient. B. Expression of ephrinA4 and CD49d (α4) on the surface of CLL cells was determined by flow cytometry (upper panels). Correlation analysis between ephrinA4 and α4 expression (mean fluorescence intensity, MFI) of samples. C. CLL cells from 30 patients (i, LApos; ii, LAneg) were preincubated (+EphA2Fc) or not (No treatment) with saturating amounts of soluble recombinant human EphA2 extracellular domains (EphA2Fc) and cultured for 12 hours in suspension in untreated plates (control) or plates containing the indicated molecules pre-immobilized to wells. BSA was used as a non-specific immobilized protein. Viability of CLL cells onto each protein is shown as percentage of basal levels in control suspension cultures without immobilized proteins. Statistical significances are shown for comparison between untreated cultures to control cultures or for EphA2Fc treatments to untreated conditions. Whisper-box plots: mean (+) and median (−) values. D. CLL cells from LApos samples (n=3) were preincubated in suspension with poly-His tagged EphA2Fc or hFc as control. Cell suspensions were then incubated with biotin anti-His followed by AlexaFluor488 streptavidin and adhered to slides for 15 min (37°C). After paraformaldehyde fixation (4% PF in PBS) slides were incubated with AlexaFluor647 conjugated monoclonal Ab for the indicated integrin chains and analyzed through confocal microscopy . At least 3 fields per experiment were evaluated for colocalization analysis (Colocalization Tool, Image J). ≥ 200 cells per sample were analyzed. E. CLL cell suspensions (10 5 /assay) were incubated for 30 min with EphA2Fc or Fc only in RPMI/2% FCS (Normal medium) or containing 1mM MnCl2 (Mn 2+ ). Next, CLL cells were incubated with a PE coupled HUTS21 mAb in cold PBS (i) or PE coupled anti-Fc preclustered VCAM-1-Fc in normal medium (ii) followed by flow cytometry analysis. A representative LApos CLL sample is shown (n>3 samples). Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques: Expressing, Flow Cytometry, Fluorescence, Recombinant, Cell Culture, Suspension, Control, Comparison, Incubation, Confocal Microscopy, Two Tailed Test

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: CLL cells of the 12 patients used in EphA2Fc assays having (LApos) or not (LAneg) lymphadenopathy (Figure ), were nucleofected with ephrinA4 specific or negative control siRNA duplexes or left untreated. A. EphrinA4 silencing was determined by flow cytometry 48 hours after nucleofection. B. Untreated or nucleofected cells (siRNA neg or ephrinA4) were left transmigrating for 12 hours through TNF-HUVEC. i) TEM rate of samples measured by flow cytometry. ii) Transwell filters from TEM assays were stained with anti CD31 mAb (green, HUVEC junctions) and Hoechst (blue, nuclei) and examined by confocal microscopy (40x oil immersion; Leica TCS-SP2). CD31 junctions were not altered in the HUVEC monolayer of ephrinA4 knock-down samples. iii) Absolute counts of CLL cells adhered to TNF-HUVEC were measured by image analysis (ImageJ; > 200 nuclei counted per filter). C. Analysis of CLL cells viability in TEM assays after ephrinA4 knock-down. i) Absolute levels of viable CLL cells in suspension cultures or in TEM assays (non-TM and TM cells) were compared between ephrinA4-specific or neg control siRNA nucleofected to control untreated CLL cells. ii) Relative viability of TM cells as percentage of basal levels in suspension cultures or of non-TM cells. Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques: Negative Control, Flow Cytometry, Staining, Confocal Microscopy, Knockdown, Suspension, Control, Two Tailed Test

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: A. Soluble ephrinA4 levels in the serum of CLL patients were determined by indirect ELISA (ng/mL) and possible associations with clinical parameters examined. B. CLL cells from 12 patients having (LApos) or not (LAneg) lymphadenopathy, used in the EphA2Fc or siRNA assays (Figures and ), were left transmigrating 12 hours through TNF-α preactivated HUVEC monolayers preincubated (30 min) with saturating amounts of recombinant human ephrinA4 (HUVEC+ephrinA4Fc), purified human Fc (HUVEC + hFc) or untreated before addition of CLL cells. i) Absolute number of transmigrated cells (TM) was counted by flow cytometry and expressed as percentage of total input cells (TEM rate). ii) Absolute levels of viable CLL cells in suspension cultures or in TEM assays (non-TM and TM cells) in the ephrinA4Fc or hFc treated TEM assays were compared to untreated conditions. iii) Relative viability of TM cells normalized to basal levels (suspension cultures) or to the corresponding non-TM cells in the ephrinA4Fc or hFc treatments compared to untreated TEM assays. C. i) Transwell filters were stained with anti VE-Cadherin (green, HUVEC junctions) and Hoechst (blue, nuclei) and examined by confocal microscopy. Adhered CLL cells were located at VE-Cadherin interendothelial junctions or separated from them. ii-iii) Caveolin-1 and ICAM-1 immunofluorescence staining of TEM assays done in chamber slides 2 hours after coculture with LApos CLL cells. ii) CLL cells inside of HUVEC were surrounded by caveolin-1 endothelial caveola (asterisks, > 1μm from VE-Cadherin junction) and ICAM-1. Insets show Z- views of caveolin and ICAM-1 staining for a a transcellular (TC) and a paracellular (PC) TEM CLL nuclei. iii) Measurement of CLL cells located within caveolin-1, at VE-Cadherin junctions or unclassified in ephrinA4Fc treated compared to untreated TNF-HUVEC after 2 hours coculture (at least 200 nuclei were counted per condition). Paired two-tailed Student's T test significance values: * P<0.05; ** P<0.01; *** P<0.001.

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques: Indirect ELISA, Recombinant, Purification, Flow Cytometry, Suspension, Staining, Confocal Microscopy, Immunofluorescence, Two Tailed Test

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: A-B. Seven μm thick sections from frozen CLL lymphadenopathies (LA n.1, 2 and 3 corresponding to CLL patients n. 3, 18 and 28, respectively) were fixed in acetone and sequentially incubated with A) TUNEL (white) and antibodies against PNAd (red) or B) antibodies against PNAd (magenta), ephrinA4 (green), EphA2 (red). In all cases nuclei were counterstained with Hoechst (blue). Fluorescent images were taken in a laser confocal microscope (TCS SP-2 AOBS, Leica). A) Representative low magnification images (20x) of areas near the capsule (“Cortical” region, left panel) or deeper (>200um far from capsule, “Inner” regions; right panel). ii) Frequency of apoptotic nuclei around (≤ 20 μm) or separated from HEV in cortical and inner LA regions (*p<0.05; **p<0.01; ***p<0.001). B) i) HEV (PNAd+, magenta; dotted line) showed strong ephrinA4 staining co-localizing with EphA2. (63x). ii) Regions of interest (ROI) were traced around HEV according to EphA2 staining (left). Intensity of ephrinA4 staining within ROIs was measured (pseudocolor scale inset). iii) HEVs containing ≥ 10% pixels above 200 gray-scales were considered as positive. Whisper-box plots of > 20 measured fields from each LA section. Unpaired two-tailed Student's t-test significance values were *p<0.01; **p<0.005; ***p<0.001. C. Human CLL cells were stained with CFSE fluorescent tracer and adoptively transferred into germ-free young Balb/c mice (2×10 7 per mice) by intravenous injection through the tail vein along with increasing concentrations of human soluble ephrinA4 purified from the sera of patients. Mice were sacrificed 24 hours later and cell suspensions from popliteal lymph nodes stained with an anti-mouse CD45 antibody, AnnexinV-PE and 7AAD for flow cytometry analysis. Human CLL cells were gated according to CFSE positivity and negativity for anti-mouse CD45 . Data (mean±SD) were compared to mice receiving human CLL cells in PBS without ephrinA4. Five animals were used per sample and experimental condition. Paired two-tailed Student's T test significance values were *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques: Incubation, TUNEL Assay, Microscopy, Staining, Two Tailed Test, Injection, Purification, Flow Cytometry

Journal: Oncotarget

Article Title: EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation

doi: 10.18632/oncotarget.10311

Figure Lengend Snippet: In patients without lymphadenopathy (LAneg, left), CLL cells have low levels of α4 integrin expression impairing their extravasation but not adhesion to endothelium likely through αLβ2. In these conditions, a low frequency of CLL cells extravasate between adjacent endothelial cells (paracellular TEM route) without receiving prosurvival signals and finally die (2). Most CLL cells, however, will detach from endothelium as a result of their high ephrinA4 expression on the cell surface allowing them to continue flowing within blood (1). The levels of soluble ephrinA4 in the sera of LAneg patients are not high enough to counteract the binding of the cell surface expressed one to endothelial EphA2 and detachment. In patients with lymphadenopathy (right) (LApos) expression of α4 integrin is high enough to allow extravasation. In these patients, CLL cell adhesion to endothelium is facilitated by low expression of ephrinA4 on their surface and the high levels of soluble isoform in sera which binds to endothelial EphA2. As a result, adhesion to endothelium will take place facilitating apoptotic signals which, nonetheless, are counteracted in the CLL cells that successfully bind to ICAM-1 and VCAM-1 induced upon EphA2 aggregation. A mechanism coupling EphA2 to caveolin-1 could promote a transcellular TEM route.

Article Snippet: Briefly, plates (MaxiSorp Nunc-Immunoplates, Nunc) were preincubated with an anti-human ephrinA4 goat polyclonal antiserum (R&D) for antigen capture followed by addition of 100 μL serum samples diluted two to eightfold in binding buffer (TBS, 0.5% Tween 20).

Techniques: Expressing, Binding Assay