primary anti-cd59 antibody  (ZenBio)

Journal: Sleep

Article Title: Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea

doi: 10.1093/sleep/zsaa286

Figure Lengend Snippet: CD59 modulates complement-stimulated vWF release from ECs. (A) Quantitation of vWF levels in HUVECs culture media in normoxia and IH after stimulation with recombinant C9 and transfection with CD59 siRNA or control RNA (ng/mL) (n = 6). (B) Representative histogram with a log scale x-axis of vWF fluorescence intensity on the cell surface in HUVECs in normoxia and IH after stimulation with C9 and transfection with CD59 siRNA or control RNA. (C) Quantitation of vWF expression on the cell surface in HUVECs after stimulation with C9 in normoxia and IH expressed as mean fluorescence intensity (n = 4). All data throughout the figure are shown as the mean ± SE two-sided t-test test. Abbreviations as in Figure 2.

Article Snippet: Primary antibodies for CD59 (R&D Systems), vWF (Abcam), syntaxin-3 (Abcam), VAMP3 and VAMP8 (Novus Biologicals), Ca v 1.2 and Ca v 3.1 (Abcam) were used.

Techniques: Quantitation Assay, Recombinant, Transfection, Control, Fluorescence, Expressing

Journal: Sleep

Article Title: Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea

doi: 10.1093/sleep/zsaa286

Figure Lengend Snippet: IH promotes co-localization of intracellular CD59 with WPB in ECs. (A) Representative confocal images of DuoLink signal between CD59 and Weibel-Palade Body (WPB) in HUVECs in normoxia and IH. EC plasma membrane is identified by immunofluorescence for VE–cadherin. Scale bar: 10 μm. (B) Quantitation of DuoLink signal in normoxia and IH (n = 4). (C) Western blot probed with antibodies directed against CD59 and vWF in the immunoprecipitate of vWF in HUVECs exposed to normoxia and IH. (D) Western blot probed with antibodies directed against CD59 and STX3 in the immunoprecipitate of STX3 in HUVECs exposed to normoxia and IH. The experiment was reproduced three times. (E) Western blot probed with antibodies directed against Cav1.2, Cav3.1, and STX3 in the immunoprecipitate of STX3 in HUVECs exposed to normoxia and IH. IgG served as negative control in (C), (D), and (E). Data in B are shown as the mean ± SE, two-sided t-test. Cav1.2, Voltage-sensitive Calcium Channel L-type 1.2; Cav3.1, Voltage-sensitive Calcium Channel T-type 3.1; STX3, Syntaxin-3. Other abbreviations as in Figure 2.

Article Snippet: Primary antibodies for CD59 (R&D Systems), vWF (Abcam), syntaxin-3 (Abcam), VAMP3 and VAMP8 (Novus Biologicals), Ca v 1.2 and Ca v 3.1 (Abcam) were used.

Techniques: Clinical Proteomics, Membrane, Immunofluorescence, Quantitation Assay, Western Blot, Negative Control

Journal: Sleep

Article Title: Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea

doi: 10.1093/sleep/zsaa286

Figure Lengend Snippet: IH enhances complement-mediated release of endothelial angiopoietin-2. (A) Quantitation of angiopoietin-2 levels in HUVEC culture media after stimulation with recombinant C9 in normoxia and IH expressed in ng/mL (n = 14). (B) Quantitation of angiopoietin-2 levels in HUVECs culture media in normoxia and IH after stimulation with C9 and transfection with CD59 siRNA or control RNA expressed in ng/mL (n = 4). All data throughout the figure are shown as the mean ± SE, two-sided t-test test. Abbreviations as in Figure 2.

Article Snippet: Primary antibodies for CD59 (R&D Systems), vWF (Abcam), syntaxin-3 (Abcam), VAMP3 and VAMP8 (Novus Biologicals), Ca v 1.2 and Ca v 3.1 (Abcam) were used.

Techniques: Quantitation Assay, Recombinant, Transfection, Control

Journal: Sleep

Article Title: Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea

doi: 10.1093/sleep/zsaa286

Figure Lengend Snippet: OSA promotes co-localization of intracellular CD59 with WPB in ECs. (A) Representative confocal images of DuoLink signal between CD59 and Weibel-Palade Body (WPB) in ECs harvested from obstructive sleep apnea (OSA) patient and control. EC plasma membrane is identified by immunofluorescence for vascular endothelial (VE)–cadherin. Scale bar: 10 μm. (B) Quantitation of DuoLink signal in OSA patients (n = 15) and controls (n = 11) (mean ± SD, permutation test).

Article Snippet: Primary antibodies for CD59 (R&D Systems), vWF (Abcam), syntaxin-3 (Abcam), VAMP3 and VAMP8 (Novus Biologicals), Ca v 1.2 and Ca v 3.1 (Abcam) were used.

Techniques: Control, Clinical Proteomics, Membrane, Immunofluorescence, Quantitation Assay

Journal: Sleep

Article Title: Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea

doi: 10.1093/sleep/zsaa286

Figure Lengend Snippet: Mechanisms mediating endothelial von Willebrand Factor and angiopoietin-2 release in OSA. Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), reduces endothelial protection against complement by reducing expression of complement inhibitor CD59 on the endothelial cell surface, which leads to increased formation and deposition of terminal complement membrane attack complex (MAC) on the cell surface. MAC promotes calcium influx resulting in increased intracellular calcium level, which enhances the releases of von Willebrand factor (vWF) and angiopoietin-2 (Ang-2) from their storage granules Weibel-Palade bodies (WPB). WPB exocytosis in IH is further augmented by binding of intracellular CD59 to syntaxin-3—a part of WPB exocytosis machinery, which leads to dissociation of syntaxin-3 from calcium channel Cav1.2 and further calcium influx. Statin stabilizes CD59 on the endothelial cell surface, and thus protects endothelium from MAC attack, which prevents release of vWF and angiopoietin-2 from WPB and may reduce pro-thrombotic and pro-inflammatory conditions in OSA.

Article Snippet: Primary antibodies for CD59 (R&D Systems), vWF (Abcam), syntaxin-3 (Abcam), VAMP3 and VAMP8 (Novus Biologicals), Ca v 1.2 and Ca v 3.1 (Abcam) were used.

Techniques: Expressing, Membrane, Binding Assay

Journal: Microbiology Spectrum

Article Title: Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death

doi: 10.1128/spectrum.02186-21

Figure Lengend Snippet: Selecting for a monoclonal HeLa CD59 KO cell line abrogates residual CD59 expression. (A) Percentages of cell death in HeLa NT (control cells, transduced with a nontargeting CRISPR sgRNA) and HeLa CD59 KO monoclonal cells when exposed to increasing concentrations of ILY, as measured by an LDH release cytotoxicity assay. The P value is <0.0001 for the points across all concentrations, as measured by two-way analysis of variance (ANOVA). The results shown are from one representative assay of >2 repeats. Each point is the mean value from 3 replicates, and error bars represent ±SD. Some error bars are contained within the point and therefore not visible. (B) Flow cytometry analysis of cell surface CD59 expression on HeLa wild type and HeLa CD59 monoclonal KO cells. The two-tailed P value is <0.0001 between the two populations, as measured by an unpaired t test. PE, phycoerythrin.

Article Snippet: Anti-CD59 (MEM-43) mouse IgG2a primary antibody (sc-51565; Santa Cruz Biotechnology) was added to the cell suspension at a dilution of 1:20, and cells were incubated for 30 min at RT in the dark.

Techniques: Expressing, Control, Transduction, CRISPR, Cytotoxicity Assay, Flow Cytometry, Two Tailed Test

Journal: PLoS Genetics

Article Title: Intermedilysin cytolytic activity depends on heparan sulfates and membrane composition

doi: 10.1371/journal.pgen.1009387

Figure Lengend Snippet: A. Resistance to ILY conferred by the knock-out of all tested genes. Values were normalized by dividing the raw IC 50 value by the IC 50 value for ILY in WT HAP-1 cells. The dashed line shows the WT IC 50 value. n = 3, error bars represent standard deviations. p-values were calculated using two-tailed t-test; *–<0.05; **–<0.01, ***–<0.001; ****–<0.0001. Significance was calculated in relation to the WT cell line. B . CD59 protein is depleted in CD59 and PIGA knock-out cell lines and significantly reduced in N-glycosylation mutant cell lines as well as TRAP complex gene ( SSR1 , SSR2 , and SSR3 ) knock-out cell lines. Cells were labeled with anti-CD59 antibody (OV9A2) conjugated with APC and FACS sorted. Median fluorescence intensity (MFI) in the APC line was compared between WT and knock-out cell lines. Values were normalized by dividing the raw MFI value of a knock-out cell line by the raw MFI value for WT HAP-1 cells. The dashed line shows the WT normalized MFI value. Error bars represent standard deviations from the mean of the medians of at least two technical replicates. Each median was calculated from at least 15000 events. p-values were calculated using two-tailed t-test; *–<0.05; **–<0.01, ***–<0.001.

Article Snippet: Subsequently, they were washed with PBS containing 1% FBS and incubated for 30 min at 37°C with a primary anti-CD59 antibody (OV9A2) conjugated with APC (Thermo Scientific).

Techniques: Knock-Out, Two Tailed Test, Glycoproteomics, Mutagenesis, Labeling, Fluorescence