Journal: Cell reports

Article Title: Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina

doi: 10.1016/j.celrep.2024.114005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Micro BCA TM Protein Assay Kit , ThermoFisher Scientific , Cat# #23235 RRID: N/A.

Techniques: Control, Affinity Purification, Recombinant, Bicinchoninic Acid Protein Assay, Western Blot, Electron Microscopy, RNAscope, Multiplex Assay, Mass Spectrometry, Mutagenesis, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: Allergic T H 2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4 + T Cells

doi: 10.3389/fimmu.2018.00750

Figure Lengend Snippet: Role of Bcl6-mediated MPT cell functions in NAT H 2 differentiation in an allergic murine model. [ (A) top] Mixture of purified KJ1-26 − MPT cells ( Bcl6 -WT or Bcl6 -KO) and KJ1-26 + WT naïve CD4 + T cells were transferred into BALB/c nu/nu mice intravenously (day 0). These mice were immunized with alum-conjugated OVA and then intratracheally challenged with OVA. [ (A) bottom] Absolute cell numbers of Neu, Eos, AM, and Lym in BALF, (B) hematoxylin and eosin-stained, formalin-fixed lung sections (magnification: 200×), and (C) T H 2 cytokine levels in the BALF of recipient mice 48 h after the last OVA challenge. (D) Relative Il4, Il5 , and Il13 expression mRNA in splenic KJ1-26 + T cells restimulated with anti-CD3 monoclonal antibodies 5 days after the last challenge. (E) OVA-specific IgE antibody titers in sera from each recipient of Bcl6 -WT NAT H 2 cells, plus MPT H 2 cells transferred from Bcl6 -TG, Bcl6 -WT, or Bcl6 -KO mice 2 days after the last challenge. All results are representative of four independent experiments with similar outcomes. Data are presented as the mean ± SEM ( n = 5–7). * P < 0.05, ** P < 0.01, comparison between two groups is indicated. AM, alveolar macrophages; BALF, bronchoalveolar lavage fluid; Bcl6, B-cell lymphoma 6; Eos, eosinophils; KJ + , KJ1-26-positive; KO, knockout; Lym, lymphocytes; MPT cell, memory phenotype CD4 + T cell; MPT H 2 cell, MPT cell-derived T H 2 cell; NAT H 2 cell; naïve CD4 + T cell-derived T H 2 cell; Neu, neutrophils; NS, not significant; OVA, ovalbumin; TG, transgenic; WT, wild-type.

Article Snippet: IgE anti-OVA Abs were detected using a mouse anti-OVA IgE Antibody Assay Kit (Chondrex, Redmond, WA, USA).

Techniques: Purification, Staining, Expressing, Knock-Out, Derivative Assay, Transgenic Assay

Journal: Frontiers in Immunology

Article Title: Circulating and Salivary Antibodies to Fusobacterium nucleatum Are Associated With Cystic Pancreatic Neoplasm Malignancy

doi: 10.3389/fimmu.2020.02003

Figure Lengend Snippet: Circulating plasma antibody reactivity to oral commensals identified in pancreatic tumor lesions. Plasma diluent (1/300) from patients with histology verified pancreas tissues: IPMN with low grade dysplasia (LGD-IPMN), IPMN with high grade dysplasia or with invasive cancer (HGD-IPMN + Cancer), or benign tumors/non-IPMN (Control) was tested in duplicates in ELISA plates coated with indicated antigen in (A–F) (bacteria: 5 × 10E7 CFU/ml, peptide 10 μg/mL). The total plasma IgG level is shown in (G) . Internal control Mean OD absorbance values of duplicates are shown. Statistical analysis was performed using the Kolmogorov–Smirnov test, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Total levels of salivary IgA and plasma IgG antibodies were measured using Human IgA/IgG ELISA Kit (Novus Biologicals, Colorado, United States) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Control, Enzyme-linked Immunosorbent Assay, Bacteria

Journal: Frontiers in Immunology

Article Title: Circulating and Salivary Antibodies to Fusobacterium nucleatum Are Associated With Cystic Pancreatic Neoplasm Malignancy

doi: 10.3389/fimmu.2020.02003

Figure Lengend Snippet: Salivary IgA antibody reactivity to oral commensals quantified by ELISA using saliva diluent (1/16) from indicated patient group. Salivary IgA reactivity to F. nucleatum , the Fap2 mimotope, and S. gordonii (A,B,F) in HG-IPMN/Cancer patients is significantly increased compared to other groups. The differences in antibody levels against bacterial antigens of G. adiacens, S. anginosus , and E. coli (C,D,E) were insignificant between groups. Total plasma IgA level did not differ between the groups (G) . Statistical analysis was performed using the Kolmogorov–Smirnov test, * p < 0.05 and ** p < 0.01.

Article Snippet: Total levels of salivary IgA and plasma IgG antibodies were measured using Human IgA/IgG ELISA Kit (Novus Biologicals, Colorado, United States) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Circulating and Salivary Antibodies to Fusobacterium nucleatum Are Associated With Cystic Pancreatic Neoplasm Malignancy

doi: 10.3389/fimmu.2020.02003

Figure Lengend Snippet: Correlation between circulating plasma (A) or salivary (B) antibody reactivity to F. nucleatum and Fap2 mimotope, or between circulating plasma antibody and salivary antibodies to either of antigen (C,D) . Competitive ELISA (E) with saliva samples with or without antigen pre-absorption as indicated for 2 h, then subjected to Fap2 mimotope ELISA test. Statistical analysis was performed using two-tailed Pearson correlation test and Wilcoxon test for two related samples. ** p < 0.01, ns = not significant.

Article Snippet: Total levels of salivary IgA and plasma IgG antibodies were measured using Human IgA/IgG ELISA Kit (Novus Biologicals, Colorado, United States) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Competitive ELISA, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 1. Vitamin D3 increases legumain expression, activity, and secretion in pre-osteoblastic cells. (A) The nucleotide sequence of the LGMN gene promoter region with annotations of potential vitamin D-responsive elements (VDRE; red) relative to the transcription start site (TSS). (B–F) Human BMSC- TERT cells (20,000 cells/cm2) were incubated with 1,25(OH)2D3 (B–F; 10, 50 or 100 nM), 25(OH)D3 (C–F; 100, 250, 500 or 1000 nM) or an equal volume of ethanol (control, 0 nM) in osteoblast induction medium for seven days before harvesting. (B) Legumain mRNA expression relative to housekeeping control (GAPDH) (2−∆∆CT; n = 3). (C) One representative immunoblot of legumain (proform 56 kDa, mature form 36 kDa) and GAPDH (housekeeping) in cell lysates (n = 3). (D) Quantification of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in C (n = 3). (E) Legumain activity (dF/s) in cell lysates adjusted for the total protein concentration (µg/mL) (n = 6–9). (F) Secreted legumain (pg/mL) in conditioned media measured by ELISA and adjusted for the total protein concentration in the corresponding cell lysates (n = 3–5). (B,D–F) Data represent mean ± SEM. (B,D) Kruskal–Wallis test. (E,F) One-way ANOVA. * p < 0.05 vs. 0 nM 1,25(OH)2D3 or 25(OH)D3. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Sequencing, Incubation, Control, Western Blot, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 2. Treatment with 25(OH)D3 increases legumain levels and activity in wild-type mice. Wild-type mice (Lgmn+/+) were treated with 50 µg/kg 25(OH)D3 (n = 7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (A) Legumain mRNA expression relative to the geometric mean of CT values of four housekeeping controls in kidney, liver, and spleen (2−∆∆CT; n = 5). (B) One representative immunoblot of legumain and GAPDH in kidney, liver, and spleen (n = 3). (C) Quantifi- cation of the 36 kDa mature legumain immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH (housekeeping) in kidney, liver, and spleen from immunoblots represented in (C) (n = 3). (D) Legumain activity (dF/s) in kidney, liver, and spleen adjusted for total protein concentration (µg/mL, n = 5). (E) Legumain plasma concentration (ng/mL) measured by ELISA (n = 5). (F) Cor- relation between legumain (ng/mL and 1,25(OH)2D3 (pmol/L) concentrations in plasma (n = 5). (A,C,E) Two-tailed unpaired Student’s t-test. (D) Mann–Whitney test. Data represent mean ± SEM. * p < 0.05. (F) Simple linear regression. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Activity Assay, Control, Injection, Expressing, Western Blot, Protein Concentration, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 3. Legumain is required for VDBP processing and regulation. (A) Purified VDBP from human plasma (1.9 µM) was incubated in legumain assay buffer (pH 5.8) at 37 ◦C with or without purified active bovine legumain (2 µM) for 5 h before gel electrophoresis and immunoblotting of VDBP (n = 1). (B–H) Wild-type (Lgmn+/+) and legumain-deficient (Lgmn−/−) mice were treated with 50 µg/kg 25(OH)D3 (n = 6–7) or an equal volume vehicle (n = 7, control) subcutaneously every two to three days (four times in total). Tissues were harvested 24 h after the final injection (day 8). (B) One representative immunoblot of VDBP and GAPDH (housekeeping) in kidney and liver (n = 4). (C–F) Quantification of VDBP immunoband (IB) intensity as arbitrary units (ARBU) relative to GAPDH in immunoblots represented in (B) (n = 4). (C) Hepatic VDBP 45 kDa immunoband. (D) Renal VDBP 45 kDa immunoband. (E) Hepatic VDBP 55 kDa immunoband. (F) Renal VDBP 55 kDa immunoband. (G) Plasma VDBP concentration (µg/mL) was measured by ELISA (n = 6–7). (H) Hepatic VDBP mRNA expression relative to the geometric mean of CT values of four house- keeping controls (2−∆∆CT, n = 5). (C–H) Data represent mean ± SEM. Two-way ANOVA. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. different genotype, same treatment. Numbers (n) represent individual biological replicates.

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Purification, Clinical Proteomics, Incubation, Nucleic Acid Electrophoresis, Western Blot, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cells

Article Title: The Cysteine Protease Legumain Is Upregulated by Vitamin D and Is a Regulator of Vitamin D Metabolism in Mice.

doi: 10.3390/cells13010036

Figure Lengend Snippet: Figure 5. Graphical representation of the suggested interplay between vitamin D and legumain. Left panel: Vitamin D (VD3) promotes legumain expression and activity through transcriptional upregulation of the legumain gene (LGMN). The free fraction of circulating VD3 metabolites diffuse through plasma membranes. 25-hydroxyvitamin D (25(OH)D3) is hydroxylated by 1α-hydroxylase (CYP27B1), forming the active metabolite 1α,25-dihydroxyvitamin D (1,25(OH)2D3). 1,25(OH)2D3 binds to the nuclear vitamin D receptor (VDR) and promotes transcription of legumain (LGMN). Synthesized prolegumain is either sorted and activated in the endolysosomal system or released to the extracellular environment. Right panel: In the proximal tubular epithelium, 25(OH)D3 bound to vitamin D binding protein (VDBP) is internalized from the tubular lumen through a megalin/cubilin- mediated process. The vitamin D metabolite is released, enabling subsequent hydroxylation by 1α-hydroxylase (CYP27B1) or 24-hydroxylase (CYP24A1), and VDBP is cleaved by legumain in the endolysosomal system. VDBP cleavage by legumain might be important in controlling the systemic level of vitamin D metabolites. Created with BioRender.com (accessed on 11 December 2023).

Article Snippet: Plasma VDBP concentrations Cells 2024, 13, 36 5 of 16 were measured using a mouse VDBP ELISA kit (R&D Systems, Catalog # DY4188-05, RRID: AB_2943630).

Techniques: Expressing, Activity Assay, Clinical Proteomics, Synthesized, Binding Assay