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: Foods

Article Title: Sea Buckthorn Pericarp Flavonoids Improve Diet-Induced Hyperlipidemia via Coordinated Modulation of Hepatic Lipid Metabolism and Gut Microbiota

doi: 10.3390/foods15061049

Figure Lengend Snippet: TFSP modulates the expression of ACC, FAS, CPT-1α, PPARα and ATGL proteins in the livers of HFD mice. n = 6 per group. Data are presented as mean ± SD. Different letters above bars indicate statistically significant differences ( p < 0.05) by one-way ANOVA followed by LSD post hoc test.

Article Snippet: Peroxisome proliferator-activated receptor alpha (PPARα) primary antibody (Cat. No. A00600-2), carnitine palmitoyltransferase-1 alpha (CPT-1α) primary antibody (Cat. No. A00917-3), acetyl-CoA carboxylase (ACC) primary antibody (Cat. No. M01802-2), fatty acid synthase (FAS) primary antibody (Cat. No. BA0484), adipose triglyceride lipase (ATGL) primary antibody (Cat. No. A01800-1), and GAPDH primary antibody (Cat. No. BM1623) were obtained from BOSTER Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing

Journal: bioRxiv

Article Title: A selective alternative pathway complement inhibitor for treatment of paroxysmal nocturnal hemoglobinuria

doi: 10.1101/2024.06.23.600249

Figure Lengend Snippet: Contrary to the antigen-driven CP and LP, the AP is initiated spontaneously by hydrolysis of C3 (“tick-over”), subsequently forming the AP fluid phase C3 convertase. Newly generated C3b is deposited on pathogen surfaces where it nucleates formation of the AP C3 convertase, amplifying the complement activity. Binding of an additional C3b molecule to the C3 convertase forms the C5 convertase, initiating C5 cleavage and thereby progression to the terminal pathway, culminating in the formation of the MAC. On healthy erythrocytes, CD55 inhibits formation and decreases stability of the C3 convertase of AP and CP/LP. For simplicity, the inhibition of the CP/LP C3 convertase is only shown illustratively and a visualization of accelerated decay of the convertases has been omitted. CD59 exerts its inhibitory function by preventing incorporation of MAC components C8 and C9. On PNH erythrocytes devoid of CD55, C3b is readily deposited, and the complement activity is amplified, resulting in progression to the terminal pathway. A lack of CD59 fails to prevent formation of the MAC, resulting in hemolysis. Abbreviations: RBC - red blood cell, Ba - factor B fragment a, Bb - factor B fragment b, fD – factor D, MAC – membrane attack complex.

Article Snippet: Following the incubation period (37°C, 1 hour), RBCs were washed twice with ice cold RBC staining buffer (PBS supplemented with 0.2% BSA) before staining on ice with anti-CD59 AF405 monoclonal antibody (FAB1987V, R&D Systems, 5 ng/μL final concentration) and anti-C3c-FITC polyclonal antibody (ab4212, Abcam, 1:300 final) for 1 h. After the staining, unbound antibodies were removed by washing the erythrocytes with RBC staining buffer twice.

Techniques: Generated, Activity Assay, Binding Assay, Inhibition, Amplification, Membrane

Journal: bioRxiv

Article Title: A selective alternative pathway complement inhibitor for treatment of paroxysmal nocturnal hemoglobinuria

doi: 10.1101/2024.06.23.600249

Figure Lengend Snippet: Flow cytometry analysis of hemolysis and C3b deposition on PNH patient erythrocytes. Two-parameter density plots, divided into four quadrants, illustrate presence of CD59 and C3c on single, intact RBCs. Normal cells (CD59 + ) are shown in Q1+Q2, PNH phenotype cells (CD59 - ) in Q3+Q4. C3b-positive cells are found in Q2+Q4. The percentages of RBCs in each quadrant are indicated. Incubation in acidified human serum (aNHS, triggering AP activation) leads to lysis of most CD59 - cells. Hemolysis of erythrocytes incubated in presence of SH-01 and eculizumab is effectively reduced to baseline levels.

Article Snippet: Following the incubation period (37°C, 1 hour), RBCs were washed twice with ice cold RBC staining buffer (PBS supplemented with 0.2% BSA) before staining on ice with anti-CD59 AF405 monoclonal antibody (FAB1987V, R&D Systems, 5 ng/μL final concentration) and anti-C3c-FITC polyclonal antibody (ab4212, Abcam, 1:300 final) for 1 h. After the staining, unbound antibodies were removed by washing the erythrocytes with RBC staining buffer twice.

Techniques: Flow Cytometry, Incubation, Activation Assay, Lysis