Journal:

Article Title: Binding of anticardiolipin antibodies to protein C via ? 2 -glycoprotein I (? 2 -GPI): a possible mechanism in the inhibitory effect of antiphospholipid antibodies on the protein C system

doi: 10.1046/j.1365-2249.1998.00582.x

Figure Lengend Snippet: Binding of protein C, phospholipids and β2-GPI. (a) Binding of protein C to phospholipids coated on the plates (□), and binding of β2-GPI to protein C (▪) in the presence of phospholipids. Protein C bound to coated cardiolipin (CL), phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). However, the binding of β2-GPI to protein C depended on the presence of anionic phospholipids (CL and PS) but not neutral phospholipids (PC and PE). Bound protein C and bound β2-GPI were detected by rabbit polyclonal anti-protein C antisera or anti-β2-GPI antisera, respectively. (b) Binding of biotinylated β2-GPI to protein C. Biotinylated β2-GPI bound to protein C in the presence of CL, but not in its absence.

Article Snippet: Bound β 2 -GPI was detected by rabbit polyclonal anti-human β 2 -GPI sera (Diagnostica Stago, Asnieres, France), followed by ALP-conjugated anti-rabbit immunoglobulin and the substrate.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids

doi: 10.1371/journal.pone.0201009

Figure Lengend Snippet: Comparison of 6 μm microcapsules and 2.35 μm PS beads for binding of antibody and for the detection of a protein analyte: (A+B) Detection of the capture antibody, BBM.1, on microcapsules and beads with fluorescently labeled goat anti-mouse antibody (GαM-AF488) by flow cytometry. (A) shows a representative experiment, (B) the average of two experiments with standard deviations (SD). (C) Schematics for the detection of human beta-2 microglobulin (hβ 2 m). The capture antibody, BBM.1, is immobilized on the protein A-coated microcapsules/PS beads. BBM.1 antibody binds specifically to hβ 2 m, which is sandwiched by the polyclonal rabbit anti-hβ 2 M (Rαhβ 2 M) antibody. The sandwich is then detected by adding AF488 labeled goat anti-rabbit (GαR-AF488) antibody. (D) Detection of hβ 2 m in PBS. Dose-response curves for the assay performed as in (C) with microcapsules or PS beads. MFI values are normalized to the maximum values. Error bars are SD (n = 3). Invisible error bars are smaller than the size of the marker. (E) Control samples of hβ 2 m plotted as histograms. Experiments were performed as in (C). Samples with analyte (10 5 pg mL -1 for microcapsules and 10 6 pg mL -1 for PS beads) were used as positive control and for normalization, which was done individually for microcapsules and PS beads. Error bars are SD (n = 3).

Article Snippet: Polyclonal goat anti-mouse antibody (Cat. No. A11001) and goat anti-rabbit antibody (Cat. No. A11008) labeled with Alexa Fluor 488 were purchased from Invitrogen, and polyclonal rabbit anti-human β 2 m (Batch 5511) was purchased from Nordic Immunology.

Techniques: Binding Assay, Labeling, Flow Cytometry, Marker, Positive Control

Journal: The FASEB Journal

Article Title: T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

doi: 10.1096/fj.12-206060

Figure Lengend Snippet: Flow cytometric immunophenotyping and Western blot analysis of the autophagy marker LC3-II in freshly isolated T cells from patients with SLE and from healthy donors. A) Flow cytometry analysis of distribution of T-cell subsets. Data are represented as box plots (white and gray box plots for healthy donors and patients with SLE, respectively) displaying medians, 25th and 75th percentiles as boxes, and 10th and 90th percentiles as whiskers. *P < 0.05 vs. normal cells. B) LC3-II Western blot analysis of T-cell lysates (30 μg/lane) from 6 healthy donors and from 6 patients with SLE. Blots shown are representative of independent experiments performed in T cells from healthy donors (n=34) and from patients with SLE (n=34). Quantification of LC3-II levels relative to β-actin in normal and SLE T cells is also shown (mean with range is presented). C) LC3-II Western blot analysis of cell lysates obtained from purified CD4+ and CD8+ naive and memory T lymphocytes. Blots shown are representative of independent experiments performed in T cells from 10 healthy donors and from 10 patients with SLE, arbitrarily chosen as representative of the whole series. Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. *P < 0.05.

Article Snippet: To ensure the presence of equal amounts of protein, the membranes were reprobed with a rabbit anti-human β-actin Ab (Amersham, Gent, Belgium) or a rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (Sigma-Aldrich).

Techniques: Western Blot, Marker, Isolation, Flow Cytometry, Purification

Journal: The FASEB Journal

Article Title: T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

doi: 10.1096/fj.12-206060

Figure Lengend Snippet: Effects of sera from patients with SLE on T-lymphocyte autophagy and apoptosis. A) LC3-II Western blot analysis of T-cell lysates (30 μg/lane) from one representative healthy donor (of the 34 analyzed). Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. Where indicated, cells were treated with the lysosomal inhibitors E64d and pepstatin A (Pep A). Statistically significant differences are indicated. B) LC3-II Western blot analysis of T-cell lysates from 3 representative patients with SLE (of the 34 analyzed). Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. C) Flow cytometry analysis of lymphocyte apoptosis. Data are referred to AV-positive apoptotic cells and are expressed as the mean ± sd of independent experiments performed in T cells from patients with SLE (n=34) and from healthy donors (n=34). Representative dot plots of flow cytometry analysis (PI on y axis vs. AV on x axis) are also shown. Numbers reported in bottom and top right quadrants represent percentages of AV single-positive cells and AV/PI double-positive cells, respectively. D) LC3-II Western blot analysis of T-cell lysates from one representative healthy donor (of 10 analyzed) after treatment with normal serum, IgG from serum of patients with SLE (50 μg/ml), serum of patients with SLE without IgG, and IVIG (50 μg/ml). Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. Statistically significant differences are indicated in the figure. E) Reactivity of human IgG purified from healthy donors (dotted line) and from sera of patients with SLE (black line) on lymphocyte surface. A representative histogram plot of flow cytometry analysis is shown.

Article Snippet: To ensure the presence of equal amounts of protein, the membranes were reprobed with a rabbit anti-human β-actin Ab (Amersham, Gent, Belgium) or a rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (Sigma-Aldrich).

Techniques: Western Blot, Flow Cytometry, Purification

Journal: The FASEB Journal

Article Title: T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy

doi: 10.1096/fj.12-206060

Figure Lengend Snippet: Susceptibility of T lymphocytes to proautophagic stimuli. A) LC3-II Western blot analysis of T-cell lysates (30 μg/lane) from one healthy donor and from one patient with SLE after treatment with 10 or 1% FBS for 4 h. Blots shown are representative of independent experiments performed in T cells from 10 healthy donors and from 10 patients with SLE, arbitrarily chosen as representative of the whole series. Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. Statistically significant differences are indicated. B) LC3-II Western blot analysis of T-cell lysates from one healthy donor and from one patient with RA after treatment with normal serum or RA serum for 48 h. Blots shown are representative of independent experiments performed in T cells from 5 healthy donors and from 5 patients with RA. Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. Statistically significant differences are indicated in the figure. C) LC3-II Western blot analysis of T-cell lysates from one healthy donor and from one patient with RA after treatment with 10 or 1% FBS for 4 h. Blots shown are representative of independent experiments performed in T cells from 5 healthy donors and from 5 patients with RA. Densitometry analysis of LC3-II levels relative to β-actin is also shown. Values are expressed as means ± sd. Statistically significant differences are indicated.

Article Snippet: To ensure the presence of equal amounts of protein, the membranes were reprobed with a rabbit anti-human β-actin Ab (Amersham, Gent, Belgium) or a rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Ab (Sigma-Aldrich).

Techniques: Western Blot

Journal: Proteomics

Article Title: Mass Spectrometric Immunoassay for Quantitative Determination of Transthyretin and its Variants

doi: 10.1002/pmic.201100023

Figure Lengend Snippet: (a) Representative transthyretin standards mass spectra, and (b) Standard curve generated with the transthyretin (TTR) mass spectrometric immunoassay by using beta-lactoglobulin (BL) as an internal reference standard.

Article Snippet: Rabbit anti-human polyclonal antibody to beta-lactoglobulin (BL) was obtained from GeneTex (Irvine, CA, Cat. No. GTX77272, 1 mg/mL).

Techniques: Generated