Journal: Cellular immunology

Article Title: Differential recognition and cytokine induction by the peptidorhamnomannan from Sporothrix brasiliensis and S. Schenckii.

doi: 10.1016/j.cellimm.2022.104555

Figure Lengend Snippet: Fig. 3. C-type lectin, TLRs receptors and signalling pathways involved in the IL-1β production by S. schenckii, S. brasiliensis and their respective pepti dorhamnomannans. PBMCs were pre-incubated for 1 h with A, B. 10 µg/ml anti-dectin-1 (n = 6), anti-dectin-2 (n = 7) and anti-mincle (n = 4) antibodies, or control isotypes IgG2b (n = 6) and IgG1 (n = 7). For assessing the signalling pathways, the cells were pre-incubated with 50 nM Syk (n = 8), 1 µM Raf-1 (n = 8) and Vehicle (DMSO; n = 8) as a control. C, D. anti-CR3 (n = 8) and anti-MMR (n = 4) antibodies, or isotype Goat IgG (n = 8) as a control, E, F. anti-TLR2 (n = 8) and 20 ng/ml Bartonella quintana LPS (n = 8), or isotype IgA2 (n = 8) and without Bart (RPMI only; n = 8) as a control. After the blocking period, cells were stimulated with heat-killed S. schenckii and S. brasiliensis or S.s PRM and S.b PRM. After 24 h of stimulation, supernatants were collected. IL-1β production was measured by ELISA. The data were expressed as mean ± SEM. Statistical analysis was performed by Wilcoxon test. *p < 0.05, **p < 0.01, ***p < 0,001, ****p < 0.0001; differs from controls of isotype antibody or Vehicle. Schematic overview of the receptors and signalling pathways required in the recognition of G. S. schenckii and S. brasiliensis, H. S.s PRM and S.b PRM.

Article Snippet: Pattern recognition receptors were inhibited with 20 ng/ml Bartonella quintana LPS, 10 μg/ml of antidectin-1, anti-dectin-2, anti-Mincle, anti-CR3, anti-MMR and antiTLR2 antibodies with their respective isotype control antibodies IgG2b, IgG1, Goat IgG and IgA2 (R&D Systems).

Techniques: Incubation, Control, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: Analysis of Tn/STn content of IgA1 from plasma: A, Total IgA plasma level (mg/ml) of IgAN patients and healthy control individuals, determined by ELISA (**, p < 0. 01). B, Western blot analysis of plasma samples after HPA-bead pull down, immunoblotted with anti-human IgA1 (α1 chain specific) antibody. All samples were treated with (+) or without (−) neuraminidase prior to HPA chromatography. Results of 14 IgAN patient samples (labeled P) are shown in the two sets of upper panels, where the top panels show the HPA+ fractions and the bottom panels show the HPA− fractions. Results of 14 control samples (labeled C) are shown in the two sets of lower panels, where the top panels show the HPA+ fractions and the bottom panels show the HPA− fractions. C, Western blot analysis of plasma samples after HPA-bead pull down, immunoblotted with anti-human IgA1 (α1 chain specific) antibody. All samples were treated with neuraminidase prior to experiments. An equal volume of plasma sample was used as the input (I) and used for HPA pull down experiments. All subsequent unbound (UB) and bound (B) fractions were loaded into the gel for analysis. D, Quantification of HPA+-IgA1 staining (B) and total IgA1 (I). All observable bands in I and B fractions were quantified. The HPA+-IgA1 fraction intensity is expressed as a percentage of the IgA1 in the corresponding input fraction. The difference of HPA+-IgA1 percentage between the IgAN patients and control samples was NS. Vertical black lines between Western blot lanes indicate that lanes were not contiguous on the same gel or from different gels. ELISA assays were performed in duplicate, twice independently.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Chromatography, Labeling, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: PNA and HPA reactivity of plasma-purified IgA1: A, B, Quantification of relative intensity of PNA and HPA, respectively, versus the IgA1 input. For each measurement, PNA and HPA intensity has been normalized by its IgA1 intensity. Lectin blots and each quantification were performed three independent times. Error bars represent ± 1 S.D. C, D, Lectin and Western blots analysis of IgAN patient and control individual samples, respectively. Plasma-purified IgA (1.5 μg) was blotted with PNA, HPA, and anti-human IgA1 (α1 chain specific) antibody (the HPA membrane was stripped and reused). E, F, Quantification of relative staining of PNA and HPA, respectively. Each individual PNA and HPA intensity has been normalized by its corresponding IgA1 intensity. No significant difference was found between the two populations. Vertical black lines between Western blot lanes indicate that lanes were not contiguous on the same gel or from different gels.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Purification, Western Blot, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: Separation of IgA1 glycoforms by lectin-based chromatography: A, Scheme of the serial HPA and PNA chromatography assays used to separate IgA1 glycoforms. B, Unbound fractions after the first round of HPA pull-down were reused for a second round of HPA pull-down and then analyzed by Western blot with anti-IgA antibody. C, Western blot analysis of the stripped membranes used in B for P2 and C19 immunoblotted with anti-human IgA2 (α2 chain specific) antibody. D, Western blot analysis after HPA pull-down followed by PNA pull-down using anti-human IgA1 (α1 chain specific) antibody. Eight IgAN patient samples are presented in the top panels (P), and 8 control samples are presented in the bottom panels (C). For B “UB” refers to the Unbound fraction, and “B” refers to the Bound fraction. For C and D “U,U” refers to the HPA−/PNA− fraction, “U,B” refers to the HPA−/PNA+ fraction, “B,U” refers to the HPA+/PNA− fraction, and “B,B” refers to the HPA+/PNA+ fraction. Vertical black lines between Western blot lanes indicate that lanes were not contiguous on the same gel or from different gels.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Chromatography, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: Reactivity of IgA1-hinge region Tn specific antibody and conversion of Tn antigen after incubation with T-synthase: A, Western blot analysis of different samples with the mouse 6E5–7Tn antibody. Arrowhead indicates the IgA1 α chain size. Neu indicates neuraminidase. B, Western blot analysis. The mouse 6E5–7Tn antibody was incubated with or without 20 mm GalNAc or with or without 2.5 μg/ml of synthetic hinge region (HR) glycopeptide containing 4 or more Tn antigens. Arrowheads indicate the IgA1 α chain signal (left gel) and the absence of signal (right gel). C, Diagram of the reaction converting Tn-containing IgA1 among total IgA1 population (IgA1-Tn/T) to T-containing IgA1 only (IgA1-T) using recombinant T-synthase in vitro. D, Western bot analyses of samples incubated with T-synthase blotted with HPA and PNA lectins, and 6E5–7Tn and anti-human IgA (α chain specific) antibodies. Healthy control sample C16 and IgAN patient sample P3 were treated with guanidinium hydrochloride (Gdm-HCl) prior to incubation with T-synthase with or without the donor substrate UDP-Gal. * indicates nonspecific binding of the goat anti-mouse HRP conjugated antibody.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Incubation, Western Blot, Recombinant, In Vitro, Binding Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: Characterization of IgA1 produced by the B cell line Dakiki: A, Semi-quantitative PCR analysis of Cosmc, T-synthase, ST6GalNAcI, and ST6GalNAcII genes in Dakiki and Tn4 cells. B, Western blot analysis of total protein cell extracts from Dakiki and the Cosmc-silenced Tn4 cells (29) blotted with HPA and PNA lectins. Protein extracts were treated with or without neuraminidase (+ or − Neu) prior to experiments. C, PNA, HPA, and IgA1 staining of 1.5 μg of media-purified IgA from Dakiki cells and plasma-purified IgA from Control and IgAN patient samples. D, PNA and HPA measured intensities relative to IgA1 intensity, from C for Dakiki and Fig. 2C, ​,22D for IgAN patients and controls. E, Western blot analysis of IgA1 after PNA (+ or − Lactose) and HPA chromatography of 1 μg of neuraminidase-treated IgA1 purified from Dakiki cell media. UB represents the unbound fractions, B represent the bound fractions. Error bars represent S.D.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Produced, Real-time Polymerase Chain Reaction, Western Blot, Staining, Purification, Chromatography

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: MS analysis of IgA1 glycoforms O-glycans: A, MS analysis of the N-glycans and B, O-glycans of purified IgA1 from plasma of control individuals and IgAN patients, and Dakiki cell culture media. C, Western blot analysis with anti-IgA antibody of HPA Unbound (UB, HPA−) and HPA Bound (B, HPA+) fractions following HPA chromatography of pooled IgAN patients and control plasma. D, MS analysis of the O-glycans of HPA+ and HPA− fractions of plasma IgA1 from controls and IgAN patients. E, Western blot analysis with the anti-IgA antibody of the HPA+ fraction of C12 sample IgA1 after incubation with T-synthase, with or without UDP-Gal. F, MS analysis of the O-glycans of the HPA+ fraction of C12 sample IgA1 after incubation with T-synthase, with or without UDP-Gal.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques: Purification, Cell Culture, Western Blot, Chromatography, Incubation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Distinct Glycoforms of IgA1 in Plasma from Patients with Immunoglobulin A (IgA) Nephropathy and Healthy Individuals *

doi: 10.1074/mcp.M114.039693

Figure Lengend Snippet: Two distinct glycoforms of IgA1 in human plasma: IgA1 is the major O-glycoprotein in human plasma. From this study, results have shown that two distinct glycoforms of IgA1 occur in plasma, the majority of IgA1 (80∼85%) with the normal O-glycans, for example, mono- and/or di-sialyl Core 1 structures which is recognized by PNA but not HPA (PNA+HPA−) after desialylation; the other, a minority of IgA1 (15∼20%) containing the Tn and STn antigens which can be bound by HPA, but not PNA (PNA−HPA+) after desialylation.

Article Snippet: IgA1-Hinge Region Specific-Tn Antibody Western Blot Mouse anti-human IgA1 (α1 chain specific) FITC-conjugated and mouse anti-human IgA2 (α2 chain specific) FITC-conjugated were purchased from (Southern Biotech, Birmingham, AL).

Techniques:

Journal: Frontiers in Medicine

Article Title: Predicting cardiometabolic disease in medical students using FibroScan and 30-year Framingham risk scores

doi: 10.3389/fmed.2024.1431935

Figure Lengend Snippet: Comparison of participants with and without elevated liver stiffness.

Article Snippet: To measure human immunoglobulin A2 and human PIGR levels, we used a commercially available enzyme-linked immunosorbent assay kit from Elabscience Biotechnology Inc., United States.

Techniques: Comparison, Control, Clinical Proteomics