l4b recombinant mouse lrpap1 protein r d systems  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells

doi: 10.3389/fimmu.2022.975680

Figure Lengend Snippet: Blocking endocytosis receptors during preincubation of APCs modulates presentation of FVIII-derived peptides and subsequent stimulation of FVIII peptide-specific CD4+ T cells hybridoma clones. APCs were preincubated with specific blocking agents for endocytosis receptors which are associated with the endocytosis of FVIII: 5mM Galactose to block the asialoglycoprotein receptors (ASGPR); 100µg/mL Heparin to block heparan sulfate proteoglycans (HSGP), 2mg/mL Mannan or 10µg/mL of a specific monoclonal antibody to block the Mannose Receptor, 14.4 µg/mL recombinant mouse LRPAP1 to block the low-density lipoprotein receptor–related protein-1 (LRP1), 1.25mM and 5mM EDTA to inhibit the function of bivalent ion-dependent receptors. After 3hrs incubation, APCs were washed and subsequently cocultured with FVIII specific CD4+ T cell hybridoma clones covering the 9 CD4 T-cell epitopes recognized by the hybridoma library. Relative IL-2 release as an indicator for the activation of hybridoma clones in relation to the normal FVIII control (100%) is presented. Depicted is the mean value derived from 2 independent experiments done in triplicates (n=6). Detailed data and statistical analysis are depicted in Table 3 .

Article Snippet: The following blocking agents were used as indicated: 5mM Galactose, 100µg/mL Heparin, 2mg/mL Mannan (all three: Sigma Aldrich, Missouri USA), 10µg/mL blocking monoclonal antibody against Mannose Receptor (Abcam, UK), 14.4 µg/mL recombinant mouse LRPAP1 (Sino Biological, China), 1.25mM and 5mM EDTA (Amresco, Ohio USA).

Techniques: Blocking Assay, Derivative Assay, Clone Assay, Recombinant, Incubation, Activation Assay

Journal: Frontiers in Immunology

Article Title: Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells

doi: 10.3389/fimmu.2022.975680

Figure Lengend Snippet: Influence of different blocking agents for endocytosis receptors present during preincubation of APCs with FVIII, on the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones .

Article Snippet: The following blocking agents were used as indicated: 5mM Galactose, 100µg/mL Heparin, 2mg/mL Mannan (all three: Sigma Aldrich, Missouri USA), 10µg/mL blocking monoclonal antibody against Mannose Receptor (Abcam, UK), 14.4 µg/mL recombinant mouse LRPAP1 (Sino Biological, China), 1.25mM and 5mM EDTA (Amresco, Ohio USA).

Techniques: Blocking Assay, Clone Assay

Journal: JCI Insight

Article Title: Monocyte-derived alveolar macrophage apolipoprotein E participates in pulmonary fibrosis resolution

doi: 10.1172/jci.insight.134539

Figure Lengend Snippet: (A and B) FITC-conjugated type I Collagen or albumin (50 μg/mL) was preincubated with conditioned media with or without mouse ApoE. The mixtures were then incubated with mouse alveolar macrophages for 4 hours. After extensive wash, flow cytometry was performed in the presence of 0.02% trypan blue. Cells without incubation with Collagen I were used as a negative control (neg.) (A). Collagen I or albumin uptake was reflected by mean fluorescence index (MFI) (B). n = 3; mean ± SD; **P < 0.01 by 2-tailed Student’s t test. (C) Six-week-old WT and ApoE–/– male mice were i.t. instilled with BLM. Four weeks later, mice were i.t. injected with 50 μg FITC-conjugated type I Collagen. Two hours later, AMs were isolated, and representative images of the AMs demonstrating Collagen I uptake are shown. Original magnification, ×200. Scale bars: 100 μm. (D) Quantitation of single cell fluorescence intensity was performed by ImageJ with a minimum of 200 cells analyzed and average calculated. Relative levels of Collagen I uptake are shown. ***P < 0.001 by 2-tailed Student’s t test. (E) Alveolar macrophages were preincubated with 2 μg/mL BSA, COG133, or LRPAP1 for 30 minutes. The cells were then incubated with conditioned media with or without ApoE for 30 minutes. After extensive wash, cell surface–bound ApoE was determined by flow cytometry. n = 3 for each group; mean ± SD. (F) A 96-well high-binding plate was precoated with 10 μg/mL Collagen I overnight at 4°C. Wells were washed and incubated with 50 ng/mL BSA or 50 ng/mL ApoE together with the indicated amounts of COG133 or LRPAP1, followed by incubation with ApoE antibody and HRP-conjugated secondary antibody and development with TMB substrate. n = 3; mean ± SD. (G) Alveolar macrophages were preincubated with 2 μg/mL BSA, COG133, or LRPAP1 for 30 minutes. The cells were then incubated with mixture of type I Collagen with control or ApoE conditioned media for 4 hours, followed by flow cytometry as in A and B. n = 3; mean ± SD. (H and I) Alveolar macrophages were treated as in G. Original magnification, ×400. Scale bars: 50 μm (H). Quantitation of single cell fluorescence intensity was performed by ImageJ and average calculated. Relative levels of Collagen I uptake are shown (I). *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Bonferroni’s post hoc test (E–I). The box-and-whisker plots depict the 25th and 75th percentiles and median, minimum, and maximum values (D and I).

Article Snippet: Recombinant mouse LRPAP1 protein was from R&D Systems.

Techniques: Incubation, Flow Cytometry, Negative Control, Fluorescence, Injection, Isolation, Quantitation Assay, Binding Assay, Control, Whisker Assay