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: Cells

Article Title: Complement Proteins C5/C5a, Cathepsin D and Prolactin in Chondrocytes: A Possible Crosstalk in the Pathogenesis of Osteoarthritis

doi: 10.3390/cells11071134

Figure Lengend Snippet: List of applied antibodies for the immunofluorescence staining.

Article Snippet: CD59 , Bio-Rad, Feldkirchen, Germany , Mouse-anti-human , MCA1054GA.

Techniques: Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

doi: 10.1242/jcs.062950

Figure Lengend Snippet: Fig. 1. Effect of Sec24 isoform silencing on transport of the endogenous GPI-anchored protein CD59 in HeLa cells. (A)Cells were transfected with siRNA against Sec24A, Sec24B, Sec24C and Sec24D, either individually or in different combinations as indicated. After 3 days, the cells were pulsed with [35S]methionine-cysteine for 10 minutes, chased for 30 minutes, and immunoprecipitated with an antibody against CD59. Immunoprecipitates were treated with (+) or without (–) endo-H and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms are indicated on the right and molecular masses (in kDa) on the left margin. (B)Quantification of endo-H resistance of CD59 from the pulse-chase experiments in A (means ± s.d., n3 independent experiments). un, untransfected; ctl, control siRNA-treated. *Statistically significant difference from untransfected cells (P<0.05; Student’s t-test).

Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

Techniques: Transfection, Immunoprecipitation, SDS Page, Autoradiography, Pulse Chase, Control

Journal: Journal of cell science

Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

doi: 10.1242/jcs.062950

Figure Lengend Snippet: Fig. 3. CD59 interacts with p24 and p23. HeLa cells were either untransfected (–) or transfected (+) with Myc-p23 (A,B,E), Myc- p23 and CD59-GFP (C), HA-p24 (D,F), HA-p24 and CD59-GFP (D), as indicated. After 48 hours the cells were subjected to immunoprecipitation (Ip), SDS-PAGE, and western blotting (Wb) using the indicated antibodies. (E,F)Cells were pulsed with [35S]methionine-cysteine for 15 minutes and subjected to immunoprecipitation with the indicated antibodies followed by SDS-PAGE and autoradiography. Note that newly synthesized Myc- p23 and HA-p24 pulled down newly synthesized endogenous CD59 and vice versa. *Bands that probably correspond to endogenous p24 and p23. IgGH, heavy chain of immunoglobulins; IgGL, light chain of immunoglobulins. Positions of molecular mass markers are indicated on the left.

Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Autoradiography, Synthesized

Journal: Journal of cell science

Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

doi: 10.1242/jcs.062950

Figure Lengend Snippet: Fig. 6. Co-partitioning of GPI-anchored proteins and p24-p23 into lipid rafts. HeLa cells were untransfected (A) or were transfected with Myc-p23 (B) or with HA-p24 (C). Cells were lysed in MBS-Triton X-100 at 4°C and analyzed by sucrose gradient centrifugation (see Materials and Methods). Fractions were collected and separated by SDS-PAGE followed by western blotting with antibodies against the indicated proteins. In parallel, the lipid raft fraction (15%+20%+25% sucrose) was collected, immunoprecipitated with anti-CD59 antibody (B and C), separated by SDS-PAGE, and immunoblotted with anti-CD59 and anti-Myc antibodies (B) or with anti-CD59 and HA antibodies (C). IgGL: light chain of immunglobulins. (D)Untransfected cells [to detect endogenous CD59, transferrin receptor (TfR), ERGIC-53, and BAP- 31] or cells transfected with Myc-p23 or HA-p24 were pulsed for 10 minutes with [35S]methionine-cysteine and subjected to sucrose density gradient centrifugation (as in A). Fractions were immunoprecipitated with antibodies against the indicated proteins or Myc and HA, and separated by SDS-PAGE followed by autoradiography. 40+endo-H, immunoprecipitated CD59 and TfR were digested with endo-H (+endo-H) before analysis. (E)Lipid raft fractions (15+20+25) from 15-minute pulse-labeled cells were pooled, immunoprecipitated with anti-HA and analyzed by SDS-PAGE followed by autoradiography. Note co-immunoprecipitation with CD59. Positions of molecular mass markers (in kDa) are shown on the left.

Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

Techniques: Transfection, Gradient Centrifugation, SDS Page, Western Blot, Immunoprecipitation, Autoradiography, Labeling

Journal: Journal of cell science

Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

doi: 10.1242/jcs.062950

Figure Lengend Snippet: Fig. 7. Cholesterol depletion by MCD disrupts the interaction of GPI- anchored proteins and p24-p23. HeLa cells either untransfected (A,C,D) or transfected with Myc-p23 or with HA-p24 (B) or with glycosylated retrieval- impaired Myc-ERGIC-53 (Myc-ERGIC-53) (C,D) were treated (+) or not (–) with 20 mM MCD for 30 minutes at 37°C. (A)Cells were lysed in MBS- Triton X-100 at 4°C, run through 5-40% sucrose gradients, subjected to SDS- PAGE and immunoblotted with antibodies against the indicated proteins. (B)Cells were lysed in 1% Triton X-100 and immunoprecipitated with anti- CD59. The immunoprecipitates were separated by SDS-PAGE and western blotting was performed with anti-CD59, anti-Myc or anti-HA. IgGL, light chain of immunoglobulins. (C,D)Cells were pulsed with [35S]methionine- cysteine for 10 minutes, chased for 30 minutes (for CD59), 40 minutes (for transferrin-receptor) or 60 minutes (for Myc-ERGIC-53), lysed in 1% Triton X-100, and subjected to immunoprecipitation with antibodies against CD59, Myc or transferrin receptor. Immunoprecipitates were treated with endo-H (+) and separated by SDS-PAGE followed by autoradiography. Endo-H-resistant (arrow) and endo-H-sensitive (arrowhead) forms of CD59, transferrin receptor (TfR) and Myc-ERGIC-53 (Myc-ERGIC-53) are indicated. MCD, methyl-– cyclodextrin.

Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

Techniques: Transfection, SDS Page, Immunoprecipitation, Western Blot, Autoradiography

Journal: Journal of cell science

Article Title: Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

doi: 10.1242/jcs.062950

Figure Lengend Snippet: Fig. 8. Effect of cholesterol depletion by MCD on the localization of endogenous CD59, ERGIC-53, p24 and Sec24C. HeLa cells were untreated (A-D) or treated (E-H) with 20 mM MCD for 30 minutes at 37°C and analyzed by immunofluorescence microscopy using antibodies against CD59, ERGIC-53 p24 and Sec24C. Note that CD59 and p24 localize to the ER in MCD-treated cells, whereas ERGIC-53 does not redistribute to the ER. Scale bar: 10m.

Article Snippet: Antibodies, siRNAs and cDNAs Mouse monoclonal antibodies: G1/93 against human ERGIC-53 (Enzo Life Sciences, Lausen, Switzerland) (Schweizer et al., 1988), G1/221/12 against human transferrin-receptor (Vollenweider et al., 1998), IgG1 against human folate receptor (Abcam, Cambridge, UK), IgG2a against human CD59 (AbD Serotec, Dusseldorf, Germany), IgG2a against human anti-tubulin (kind gift from Karl Matter, University College London, UK), A1/182/5 against human BAP31 (Enzo Life Sciences, Lausen, Switzerland), A1/118/4 against human GPP130 (Linstedt et al., 1997), 9E10.2 against the Myc epitope (ATCC #CRL 1729), 12CA5 against the hemagglutinin (HA) epitope, IgG1 against green fluorescent protein (GFP; Roche, Rotkreuz, Switzerland), anti-VSV-G (clone P5D4, kind gift from Kai Simons, Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany).

Techniques: Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: The classical pathway is triggered by interaction of C1 with immune or non-immune complexes leading to conformational changes in C1q, activation of C1r and C1s, and subsequent assembly of C3 convertase. The lectin pathway is activated by binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface, which activates the MBL-associated serine proteases (MASPs), followed by formation of C3 convertase. Spontaneous hydrolysis of C3 initiates the alternative complement pathway. All three pathways of the complement cascade converge on the classical C3 convertase, which cleaves and activates component C3, forming C3a and C3b. This triggers a series of further cleavage and activation events, leading to cleavage of C5 into C5a and C5b, and eventual formation of the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and C9. MAC is the terminal cytolytic complex of the complement pathway; it causes osmotic lysis of target cells by forming transmembrane channels that disrupt the phospholipid bilayer of target cells. The complement system is tightly regulated by the following complement control proteins: CFH, CFHR1, CFHR4, CFB, CD55, CD59, CD46, CFI, and CFP.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Activation Assay, Binding Assay, Lysis

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Markers’ Information.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Marker, Activation Assay, Lysis, Inhibition

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Genes’ Information.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Proteins’ Western Blotting Information.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Gene Expression Data.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Decreased protein levels of CFH, CFHL1, CD55, CD59, CFI, and CD46, and increased levels of CFP, CFB, CFHR4, and CFHR1 protein in AMD cybrids. (A, C, E, G, I, K, M, O, Q, S) Representative Western blots of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 respectively. (B, D, F, H, J, L, N, P, R, T) Graphs showing quantitation of CFH, CFHL1, CD55, CD59, CFI, CFP, CFB, CD46, CFHR4, and CFHR1 proteins in Older-Normal and AMD cybrids. * P < 0.05, ** P < 0.01. n = 4–5. Data were analyzed using Student’s T-test.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Differential Expression of Complement Markers in Normal and AMD Transmitochondrial Cybrids

doi: 10.1371/journal.pone.0159828

Figure Lengend Snippet: Complement Protein Expression Data.

Article Snippet: 3 , CD59 , 14 kDa (Monomer) ~ 40kDa (Glysolysated form) , Rabbit Anti-Ly6c/CD59 Polyclonal antibody bs-12327R (Bioss USA) OWL ID# 37854 (One world Lab) , 1:1000 , Human , Rabbit IgG Ab (HRP) GTX 213110–01 (Genetex) , beta-actin antibody GTX 110564 (Genetex).

Techniques: Expressing