Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: (A, B) TEM immunogold localization of sCLU in RBCs membrane protrusions (A) and vesicles (ves) (B) collected from fresh units of stored RBCs (N = 2, young healthy donors). Solid or dashed arrows indicate sCLU immunogold localization at the periphery or the cytosol of the vesicles, respectively. (C) Representative immunoblot analysis of RBCs-derived purified vesicles (N = 2) probed with either polyclonal anti-sCLU or with monoclonal anti-Band 3 antibodies. Molecular weight markers are indicated to the right of the blot. Bars in (A), (B), 100 nm.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Membrane, Western Blot, Derivative Assay, Purification, Molecular Weight

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with polyclonal antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) Anti-dinitrophenylhydrazone (DNP) immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Purification, Immunoprecipitation, Control, Clinical Proteomics, Membrane, Staining, Western Blot, Modification

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Erythrocytic sCLU localizes at both sides of the plasma membrane in association with non-cytoskeletal areas, as well as in the cytosol (see also, Antonelou et al., accompanying paper). At the intracellular side of the RBCs membrane sCLU may bind Band 3, Hb and/or other cytoskeleton-free membrane portions. On the other hand, the sCLU that localizes at the extracellular side of the RBCs membrane can attach to membrane by binding to Band 3, CD59, plasma membrane IgGs or to an currently unknown sCLU-specific receptor. Physiological in vivo or ex vivo RBCs senescence (1) is associated with cytosol, cytoskeleton and membrane structural alterations, including Band 3 modifications, increased membrane binding of IgGs, proteolysis, protein aggregation and increased oxidation defects. Vesiculation (2), a self-protective mechanism of mammalian erythrocytes, removes oxidized proteins and aggregates from both plasma membrane and cytosol thereby postponing the untimely elimination of otherwise healthy erythrocytes. This process takes place through the entire in vivo or ex vivo lifespan of RBCs and is functionally connected to the release of sCLU-, Band 3-, CD59-, Hb- and IgGs-containing vesicles. We propose that vesicular sCLU by following its membrane linkers (e.g. Band 3) or other unknown cytosolic interacting proteins assists via its chaperone function in the disposal of non-functional or death signalling effective material from RBCs.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Clinical Proteomics, Membrane, Binding Assay, In Vivo, Ex Vivo, Functional Assay

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 1. p53 binds to the putative binding sites within the CD59 gene in vitro. A, CD59.1, the first putative p53-responsive element, is located in the 5V flanking region of the CD59 gene ranging from nucleotide 1665 to 1637 upstream of exon 1. CD59.2 is the second putative p53-binding sequence located in intron 1 of CD59 ranging from nucleotide 675 to 645 upstream of exon 2. Possible p53 half-binding sites are marked in capital letters. The differences between these and the p53 consensus binding sequence are underlined. B, EMSA with samples labelled with horseradish peroxidase, incubated with 100 ng wild-type recombinant p53 in the presence of 0.1 Ag/AL poly(deoxyinosinic-deoxycytidylic acid). Oligonucleotides were separated in a 2% agarose gel and detected by autoradiography.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Binding Assay, In Vitro, Sequencing, Incubation, Recombinant, Agarose Gel Electrophoresis, Autoradiography

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 2. p53-dependent expression of CD59 in HeLa cells. A, Western blot analysis of p53 (anti-p53, cloneBP53-12 recognizes all p53 modifications) and CD59 expression in HeLa cells and cells 36 hours after transfection with siRNA specifically blocking expression of p53. Detection of p42 MAPK was done as a control for the siRNA specificity. B, fluorescence-activated cell sorting analysis of CD59 expression in HeLa and p53 siRNA–transfected cells.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Transfection, Blocking Assay, Control, Fluorescence, FACS

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 3. Alterations in CD59 expression in IMR32 cells treated with camptothecin. A, Western blot analysis of CD59 expression in IMR32 cells treated with camptothecin for either 24 or 48 hours. B, expression of CD59 in IMR32 cells treated with camptothecin for 24 or 48 hours detected by quantitative PCR. Expression in untreated cells is set as 100%. Columns, mean for two independent experiments; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison. C, resistance of IMR32 (x) and surviving IMR32 (n) cells to C lysis. Lysis assay with preincubation of both untreated (.) and surviving (E) IMR32 cells with CD59-blocking antibody BRIC229 was carried out as a control. Points, mean for three independent experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Comparison, Lysis, Blocking Assay, Control

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 4. Expression of p53 and recruitment to the CD59 gene. A, two different antibodies were used for detection of p53: anti-p53, clone BP53-12 recognizing all forms of p53 and anti–acetyl-p53 (Lys373, Lys382) to detect the changes in p53 acetylation. B, IMR32 cells were treated with camptothecin for either 24 or 48 hours. Immunoprecipitation was done with anti–acetyl-p53 (Lys373, Lys382) antibody. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (striped columns) in control IMR32 cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Control, Chromatin Immunoprecipitation, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 6. Chromatin immunoprecipitation analysis of recruitment of p53 to the CD59 gene in living Hep3B cells. Cells were treated with IFN-g or IL-8 for 36 hours. Immunoprecipitation was carried out either with anti–acetyl-p53 (Lys373, Lys382; A) or anti-p53, clone BP53-12 (B) antibodies. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (white columns) in untreated cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 5. Effect of cytokines on p53 and CD59 expression in IMR32 and Hep3B cells. A, quantitative PCR analysis of p53 (black columns) and CD59 (white columns) expression following an incubation of Hep3B and HL60 cells with IFN-g or IL-8. Results from two independent measurements displayed a significant difference between untreated and cytokine treated Hep3B samples. *, P < 0.05; **, P < 0.01. However, no significant difference was observed between HL60 samples. B, flow cytometry analysis of p53 (black columns; Lys373, Lys382) acetylated p53 (white columns) and CD59 (striped columns) following the same treatments as in (A). *, P < 0.01; **, P < 0.001. Columns, mean of four measurements obtained from two separate experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Flow Cytometry

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet: In vivo expression of eGFP, PEDF, sFlt-1, and sCD59 following IVT administration of AdCs (A) Representative fundus images of the retina from individual mice following IVT injection of 1.5×10 9 vp and 7.5×10 9 vp AdC68-eGFP. The eGFP signal could be detected from 48 h to 35 days post-injection. The dotted circles represent the edge of mouse retina. (B–D) Assessment of PEDF, sFlt-1, and sCD59 mRNA expression in retina-choroid-sclera complexes isolated from 10 mice. In each mouse, one eye was injected with AdC68-PFC (five mice for 1.5×10 9 vp and five mice for 7.5×10 9 vp) whereas the contralateral, un-injected eye served as control (only five eyes were used for analysis). At 4 days post-injection, RNA was purified from the retina-choroid complexes and real-time qPCR was conducted. Absolute number of mRNA copies were calculated using the standard curve method. (E–H) Images of western blot and quantification of the PEDF, sFlt-1, and sCD59 protein amount expressed in retina-choroid complexes of five mice. In each mouse, one eye was injected with AdC68-PFC (7.5×10 9 vp) whereas the contralateral, un-injected eye served as control. Total protein was obtained from retina-choroid-sclera complexes isolated from AdC68-PFC-treated (7.5×10 9 vp) and un-injected eyes 7 days post-injection. Antibodies against GAPDH were used for the internal control. The relative expression of PEDF, sFlt-1, and sCD59 in the un-injected eyes was set to 1. Data are expressed as mean ± SEM, and analyzed using one-way ANOVA multiple comparisons with Tukey’s method among groups in (B) and Student’s t test (two-tailed) in (C) (∗p < 0.05, ∗∗p < 0.01). PEDF, pigment epithelium-derived factor; sFlt-1, soluble fms-like tyrosine kinase-1; sCD59, soluble forms of CD59; IVT, intravitreal.

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: In Vivo, Expressing, Injection, Isolation, Control, Purification, Western Blot, Two Tailed Test, Derivative Assay

Journal: iScience

Article Title: Chimpanzee adenovirus-mediated multiple gene therapy for age-related macular degeneration

doi: 10.1016/j.isci.2023.107939

Figure Lengend Snippet:

Article Snippet: PVDF membranes were blocked with 5% milk in PBST (PBS+Tween-20) for 2 h at RT and then target proteins were detected by specific primary antibodies including PEDF (1:500, 11104-RP02, Sino Biological, China), VEGFR1 (1:500, AF7748, Affinity, US), sCD59 (1:500, 12474-RP02, Sino Biological, China), Erk1/2 p44/42 (1:1000, 9102, Cell Signaling Technology, US), Erk1/2 phospho-p44/42 (T202/Y204) (1:1000, 9106, Cell Signaling Technology, US), p38 MAPK(1:1000, 8690, Cell Signaling Technology, US), Phospho-p38 MAPK (Thr180/Tyr182) (1:1000, 4631, Cell Signaling Technology, US), ICAM-1 (1:1000, 10831-1-AP, Proteintech, US), VCAM-1 (1:200, sc-13160 , Santa Cruz biotechnology, US).

Techniques: Virus, Recombinant, Transfection, Protease Inhibitor, Plasmid Preparation, Gel Extraction, Software

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a The cartoon representations of the substrates and products of the GWT1 enzyme are displayed. The chemical structure of manogepix, an inhibitor of GWT1 protein, is demonstrated. The hydroxyl group that accepts the palmitoyl group (yellow) is highlighted in the red circle . b Topology of fGWT1 presents the specific sites of GFP insertion. Through screening, A fusion of GFP at the Y422 position construct exhibited the most homogeneous protein properties. c FACS (fluorescence-activated cell sorting) of HEK293T △PIGW cells expressing fGWT1 (light green) using CD59 as the marker with gating strategies. HEK293T WT cells are highlighted in red, while individual HEK293T △PIGW cells are marked in blue. Those HEK293T △PIGW cells responding to fGWT1 are labeled in light green. The background served as a blank control, referring to HEK293T △PIGW cells not stained with CD59 antibodies. d Fluorescent confocal microscopy analyses were conducted to assess fGWT1 activity, with the construct and cell line indicated alongside the images. The first, second, and third columns illustrate confocal fluorescence microscopy images of CD59, DAPI, and merged views, respectively. A scale bar of 10 μm is included. The confocal microscopy experiment was repeated at least three times.

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Construct, Fluorescence, FACS, Expressing, Marker, Labeling, Control, Staining, Confocal Microscopy, Activity Assay, Microscopy

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a , d The cryo-EM map of GWT1 is displayed from both side ( a ) and top ( d ) perspectives, with the luminal access cavity highlighted by a blue dashed oval. b , e An additional density within the highly conserved luminal access cavity. c Molecular lipophilicity potential (MLP) of the luminal access cavity. The surfaces are colored by lipophilicity potential calculated by Chimera X. The putative entry route for GlcN-PI is indicated by a dashed blue path. f Comparison of catalytic sites of GWT1 and HGSNAT. The predicted catalytic sites of HGSNAT are N286 and H297, while the putative catalytic sites of GWT1 are D145 and K155. g Expressing fGWT1 in HEK293T △PIGW cells restores CD59 staining in the FACS assay. HEK293T △PIGW cells, where GPI-AP biosynthesis is blocked, serve as the staining control (gray). Loss-of-function mutants are highlighted in brilliant blue. Coordination residue mutants of the luminal access cavity are highlighted in purple.

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Cryo-EM Sample Prep, Comparison, Expressing, Staining, Control, Residue

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a Previously reported drug resistance sites for GWT1 are shown as spheres and colored in gray-blue. b All drug resistance sites, including those previously reported and newly identified, are shown. c Evaluating the sensitivity of manogepix by staining CD59 in the FACS assay, expressing fGWT1 in HEK293T △PIGW cells. Cells treated with manogepix (orange) or without manogepix (blue) were analyzed by flow cytometry using gating strategies, with negative controls marked in red. All experiments were conducted independently three times ( n = 3).

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Staining, Expressing, Flow Cytometry

Journal: Nanomaterials

Article Title: A Closed-Loop Autologous Erythrocyte-Mediated Delivery Platform for Diabetic Nephropathy Therapy

doi: 10.3390/nano12203556

Figure Lengend Snippet: Changes in blood parameters during the hemodialysis process of the CAER platform. ( A ) Mean concentration changes in unencapsulated INS in the serum. ( B ) Mean concentration changes in unmodified GOx from GOx-INS@ERs. ( C , D ) Effects of hemodialysis on the INS concentration and GOx in the GOx-INS@ERs. ( E ) CREA, BUN, Ca 2+ , and K + concentrations before and after hemodialysis. ( F ) Surface expression levels of CD55 and CD59 on GOx-INS@ERs and natural ERs measured by flow cytometry. ** p < 0.01.

Article Snippet: Rabbit anti-CD55 antibody (bs-1552R), rabbit anti-CD59 antibody (bs-1638R), and mouse anti-rabbit IgM/FITC antibody (bs-0369M-FITC) were from BIOSS (Beijing, China).

Techniques: Concentration Assay, Expressing, Flow Cytometry