cd59 primary antibody  (Thermo Fisher)

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: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Role of CD59 and expression in different cancers. ( A ) involvement of CD59 in different biological pathways using ShinyGO v0.741. ( B ) Comparison of CD59 mRNA expression between cancer and its normal tissue counterpart in multiple cancers using the TCGA database and the GAPIA2 analytical tool. ( C ) mRNA expression of CD59 in KIRC, CESC, GBM, HNSC, and STAD cancer patients and normal tissue from the TCGA database using the GAPIA2 analytical tool. ( D i – v ) Protein expression of CD59 in KIRC ( i ), CESC ( ii ), GBM ( iii ), HNSC ( iv ), and STAD ( v ) cancer patients and normal tissue from the Human Protein Atlas. Each dot represents mRNA expression of sample and * indicates p ≤ 0.05.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Comparison

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Biological pathways related to the proteins involved in CD59 networks based on KEGG pathways (based on STRING database).

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Coagulation, Infection

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: List of cancers and CD59 expression with significance.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Expression of CD59 in cancer cell lines and cancer patients. ( A ) mRNA expression of CD59 in HEK 293T (normal human embryonic kidney), 786-O (KIRC), HeLa (CESC), and SF188 (GBM) by RT-PCR. ( B – D ) protein expression of CD59 in HEK 293T (normal human embryonic kidney), 786-O (KIRC), HeLa (CESC), and SF188 (GBM) by Western blot ( B ), flowcytometry (surface staining) ( Ci ), and its quantification ( Cii ), and immunofluorescence (surface staining) ( Di ) with its quantification ( Dii ). β-tubulin (control) for B (786-O, HeLa, and SF188) are similar to Figure 7A(iii). Data represents the minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Control

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Prognostic analysis of CD59 in cancer. ( A i – v ) Analysis of CD59 expression and overall survival (OS) using Kaplan–Meier in KIRC ( i ), CESC ( ii ), GBM ( iii ), HNSC ( iv ), and STAD ( v ) using the GEPIA 2 dataset. ( B ) Analysis of clinical relevance of CD59 expression across various cancer types using Timer 2.0 analytical tool.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Correlation between CD59 expression and Treg and MDSC. ( A ) Immune subtype analysis of ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD using TISIDB. ( B ) expression and distribution of CD59 on immune cells by the HPA dataset ( Bi ) and Schmiedel database ( Bii ). ( C ) Flow cytometry analysis (Ci) with quantification (Cii) of FOXP3 intracellular expression on 786-O, HeLa, and SF188 cell lines. Correlation between CD59 and Treg cells ( D ) and MDSC cells ( E ) in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD using TISIDB. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Flow Cytometry

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Correlation between CD59 expression and TAM and immune-infiltrating M2 macrophage: ( A ) Spearman correlation between CD59 and macrophage in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD. ( B ) Heatmap of correlation between CD59 and ( i ) macrophage, IL6, IL6R, and ( ii ) IL10, and IL10RB in KIRC, CESC, GBM, HNSC, and STAD. ( C ) Scattered plot of relationship between M2 macrophage infiltration and CD59 expression in ( i ) KIRC, and ( ii ) GBM, and STAD using Timer 2.0 analytical tool.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Co-culture of tumor cells with macrophage increases infiltrating M2 macrophage: ( A ) Analysis of tumor cells in the co-culture system. ( Ai ) Basic expression of phosphorylated STAT3 (Y705) in tumor cells. ( Aii ) Expression of pSTAT3 (Y705) in tumor cells in the presence or absence of macrophage. ( Aiii ) mRNA expression of IL10 in tumor cells in the presence or absence of macrophage. ( Aiv ) mRNA expression of CD59 in tumor cells in the presence or absence of macrophage. ( B ) Analysis of macrophage in the co-culture system. ( Bi ) Gating strategy to identify M2 macrophage. ( Bii ) Percentage of M2 macrophage in 786-O, HeLa, and SF188. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Co-Culture Assay, Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: TGF-β-mediated immune suppression in CESC, GBM, HNSC, and STAD. ( A ) Expression analysis of TGFβ1. ( Ai , ii ) Flow cytometry analysis of TGF-β expression on 786-O, HeLa, and SF188 cell line. ( Aiii ) Immunoblotting of TGFβ1 and ( Aiv ) ELISA of secreted TGFβ1. ( B ) Spearman correlation analysis of TGF-β and CD59 expression in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD and supported by ( Ci ) heat map of TGF-β and CD59 expression in KIRC, CESC, GBM, HNSC, and STAD using TISIDB. ( Cii ) Correlation between CD4+ and CD8+ T cell immune infiltration and CD59 expression in multiple cancers using Timer 2.0 analytical tool. β-tubulin (control) for A(iii) (786-O, HeLa, and SF188) are similar to B. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Diagram of CD59-mediated immune suppression in CESC, GBM, HNSC, and STAD. CESC, GBM, HNSC, and STAD have high amounts of FOXP3, IL10, TGFβ1, and pSTAT3, leading to increased CD59 transcription and the recruitment of immune suppressive cells such as MDSC, Treg, and TAM in the TME.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques:

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