Journal: iScience

Article Title: FoxO-KLF15 pathway switches the flow of macronutrients under the control of insulin

doi: 10.1016/j.isci.2021.103446

Figure Lengend Snippet:

Article Snippet: Rabbit control IgG , Sino biological , CR1.

Techniques: Plasmid Preparation, Recombinant, Protease Inhibitor, Lysis, Luciferase, Enzyme-linked Immunosorbent Assay, Mutagenesis, Expressing, Software

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Kinetic and equilibrium dissociation constants for the binding of C1q to immobilized soluble CR1 (sCR1) and CR1 CCP22-30.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Localization of the C1q binding sites in CR1 CCP22-30 using deletion fragments. (A) Schematic view of the different fragments of CR1 CCP22-30. Potential N-glycosylations are represented by open circles ○. (B) Interaction of C1q with immobilized CR1 CCP22-30 fragments analyzed by ELISA. C1q (10 µg/ml in PBS) was added to microtiter plate coated with 3.4 pmol of each CCP22-30 fragment as described in Section “ .” After 1.5 h incubation at room temperature, bound C1q was detected with rabbit antibodies recognizing C1q and HRP secondary antibodies. Data are presented as the mean ± SE of four individual experiments. (**), Student’s t -test values p < 0.01 of C1q binding to each fragment compared to CCP22-30.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Complement receptor type 1 (CR1) variants produced to study the CCP24-25 module pair interaction properties. (A) Schematic view of CCP24-25 variants produced in eukaryotic cells. Potential N-glycosylations are represented by open circles ○. (B) SDS-PAGE analysis of 4 µg of the CR1variants under reducing conditions. The positions of the molecular weight markers (expressed in kilodaltons) are indicated. A full scan of the original gel is provided in Figure S1 in Supplementary Material .

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Produced, SDS Page, Molecular Weight

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Kinetic analysis of the interaction of C1q and mannose-binding lectin (MBL) with CR1 CCP24-25. C1q (A) and MBL (B) , at indicated concentrations, were injected in 50 mM triethanolamine-HCl (TEA), 145 mM NaCl, 0.05% surfactant P20, pH 7.4, on immobilized CR1 CCP24-25 (1,000 RU). The buffer was supplemented with 3 mM EDTA for MBL binding. Fits are shown as dotted lines and were obtained by global fitting of the data using a 1:1 Langmuir-binding model. The kinetic constants obtained are framed on the top of each sensorgramm.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Interaction of C1q and mannose-binding lectin (MBL) with immobilized CR1 CCP22-30 and CR1 ΔCCP24-25. The binding curves were obtained in single cycle mode by injecting increasing concentrations of C1q (A) or MBL (B) on immobilized CR1 CCP22-30 (3,000 RU) or CR1 ΔCCP24-25 (2,200 RU). C1q (0.25, 0.5, 1, 2, and 4 nM) was injected at 20 µl/min for 180 s in 50 mM triethanolamine-HCl (TEA), 150 mM NaCl, 1 mM CaCl 2 , 0.05% surfractant P20, pH 7.4. MBL (1, 2, 4, 8, and 16 nM) was injected in the same conditions except that the buffer contained 3 mM EDTA instead of CaCl 2 .

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Elongated shape of CCP24-25, associated to a possible interpretative model. (A) Small-angle X-ray scattering (SAXS) pair distance distribution, (B) SAXS dimensionless Kratky plot, which shows elongation and remaining flexibility. (C) Fit of the model [shown in panels (D,E) ] to the experimental data. (D) Top and (E) side views of an ab initio envelope computed with GASBOR. A Coral-derived model of CCP24-25 is shown inside to illustrate how the shape corresponds to two CCP modules. This interpretative model is used to illustrate the positions on each module of glycosylation sites (green), rs 4844609 SNP (magenta), Knops groups variants (cyan), acidic clusters (red) initially suggested as potential MBL binding sites, and the other acidic residues (gray and black). D1529 is the homologous counterpart of D1076 in CR1 CCP17 (black). The two flexible carbohydrates included in the model are only illustrated in the side view.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Derivative Assay, Glycoproteomics, Binding Assay

Journal: Frontiers in Immunology

Article Title: C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules

doi: 10.3389/fimmu.2018.00453

Figure Lengend Snippet: Localization of the binding site of soluble CR1 on C1q. (A) Comparative binding of C1q and its CLF and GR on complement receptor type 1 (CR1) CCP22-30 analyzed by surface plasmon resonance (SPR). The CR1 CCP22-30 fragment was immobilized on CM5 sensor chips (4,500 RU) and 2 nM of C1q or CLF and 12 nM of GR were injected over the surfaces as described in Section “ .” (B) MBL-associated serine protease (MASP)-3 competition of the binding of C1q to CR1 CCP22-30 analyzed by SPR. C1q (2 nM) was incubated at room temperature for 20 min in the absence or presence of recombinant MASP-3 at indicated molar ratios and injected over immobilized CR1 CCP22-30 (4,500 RU). No binding was observed when MASP-3 alone (20 nM) was injected.

Article Snippet: Recombinant soluble human CR1 (sCR1) was purchased from R&D Systems Europe.

Techniques: Binding Assay, SPR Assay, Injection, Incubation, Recombinant

Journal: Translational Vision Science & Technology

Article Title: Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model

doi: 10.1167/tvst.7.2.3

Figure Lengend Snippet: ECT to deliver CR2-fH—tool development. (A) ARPE-19 cells encapsulated in alginate were spotted onto a glass slide for imaging; and (B) plated after dissolving the alginate wall to document viability. Cell survival in the capsules was assessed using Calcein AM (C) indicating viable cells by green fluorescence and Ethidium homodimer-1 indicating dead cells by red fluorescence. (D) Stably transfected ARPE-19 cells secrete CR2 and CR2-fH toward both the apical and basal side when grown as monolayers on transwell plates (supernatants from three different cultures).

Article Snippet: CR2-fH was detected using an anti-CR2 primary antibody (10 μg/mL; rat anti-mouse CD21, clone 7G6; purified in house 30 ) incubated in 5% nonfat milk/TBST (1:1000) overnight, and visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rat; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by incubation with ClarityTM Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.).

Techniques: Imaging, Capsules, Fluorescence, Stable Transfection, Transfection

Journal: Translational Vision Science & Technology

Article Title: Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model

doi: 10.1167/tvst.7.2.3

Figure Lengend Snippet: ECT to deliver CR2-fH—documentation in the eye. (A) Capsules can be imaged in the eye using OCT. (B) Production of CR2-fH within the capsules and diffusion of the fusion protein throughout the retina layers was confirmed by immunohistochemistry using an antibody against CR2. The corresponding DIC and fluorescence image is presented. CR2 antibody staining was negative in uninjected control eyes. (C) After intravitreal capsule delivery, CR2-fH was detectable in the RPE/choroid fraction of eyes with CNV lesions (breach of the BRB) but not in those with without. A dot blot of RPE/choroid samples with 2-fold dilution steps is presented. Representative examples from more than three independent experiments are shown.

Article Snippet: CR2-fH was detected using an anti-CR2 primary antibody (10 μg/mL; rat anti-mouse CD21, clone 7G6; purified in house 30 ) incubated in 5% nonfat milk/TBST (1:1000) overnight, and visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rat; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by incubation with ClarityTM Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.).

Techniques: Capsules, Diffusion-based Assay, Immunohistochemistry, Fluorescence, Staining, Control, Dot Blot

Journal: Translational Vision Science & Technology

Article Title: Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model

doi: 10.1167/tvst.7.2.3

Figure Lengend Snippet: CR2-fH produced in the eye does not cause an immune response. Intraocularly produced CR2-fH could potentially gain access to the circulation and lymph nodes. Lack of IgG or IgM antibody production was confirmed 1 month after capsule injection harboring ARPE-19 cells expressing CR2 or CR2-fH. Supernatants from cells expressing CR2-fH were run at two different concentrations and probed for the presence of CR2-fH using the anti-CR2 antibody to identify the size of the protein (positive control). Identical lanes were probed with serum from experimental animals (S-E; injected with CR2-fH capsules) or control animals start with (S-E; age-matched animals without injections S-C) to match the description of the experiment at 1:50, followed by appropriate secondary antibodies.

Article Snippet: CR2-fH was detected using an anti-CR2 primary antibody (10 μg/mL; rat anti-mouse CD21, clone 7G6; purified in house 30 ) incubated in 5% nonfat milk/TBST (1:1000) overnight, and visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rat; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by incubation with ClarityTM Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.).

Techniques: Produced, Injection, Expressing, Positive Control, Capsules, Control

Journal: Translational Vision Science & Technology

Article Title: Encapsulated Cell Technology-Based Delivery of a Complement Inhibitor Reduces Choroidal Neovascularization in a Mouse Model

doi: 10.1167/tvst.7.2.3

Figure Lengend Snippet: ECT-mediated delivery of CR2-fH reduces CNV and complement activation. One month following intravitreal injection of alginate capsules, laser-induced CNV was performed. Lesion spot sizes were analyzed 5 days later using OCT, complement activation using ELISA for the anaphylatoxin C3a. (A, B) CNV sizes were reduced in eyes loaded with alginate capsules containing ARPE-19 cells expressing CR2-fH as opposed to those expressing CR2, no extra cargo (native ARPE-19), or empty capsules containing media only. The three control groups did not differ from each other. (C) CNV-induced complement activation as demonstrated by elevated levels of C3a when compared to animals with no lesions (control). C3a levels were elevated in RPE/choroid fractions of animals exposed to CR2, native ARPE-19 cells, or empty capsules, and significantly reduced by CR2-fH. Data shown are average values (±SEM) (n = 3–18 animals per condition as indicated).

Article Snippet: CR2-fH was detected using an anti-CR2 primary antibody (10 μg/mL; rat anti-mouse CD21, clone 7G6; purified in house 30 ) incubated in 5% nonfat milk/TBST (1:1000) overnight, and visualized with a horseradish peroxidase-conjugated secondary antibody (anti-rat; Santa Cruz Biotechnology, Inc., Dallas, TX) followed by incubation with ClarityTM Western ECL Blotting Substrate (Bio-Rad Laboratories, Inc.).

Techniques: Activation Assay, Injection, Capsules, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Sulforaphane suppresses the growth of triple-negative breast cancer stem-like cells in vitro and in vivo

doi: 10.1158/1940-6207.CAPR-18-0241

Figure Lengend Snippet: Selected validation of Nanostring gene expression analysis by qRT-PCR. Four genes including CR1, FOXD3, WNT3, and NOTCH4 are shown here as representatives for amplification. Data are the average of 8 samples from SFN pre-treated group and 8 samples from saline control. All experiments were conducted in duplicates. *: P < 0.05 as compared to the control animals.

Article Snippet: CR1 (R&D Systems, Cat# 145-CR/CF), Nodal (R&D Systems, Cat# 3218-ND-024/CF), GRP78 (Abcam, Cat# ab78432), and Alk4 (Creative BioMart, Cat# ACVR1B-645H,) were purchased from the indicated vendors.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Amplification, Saline, Control

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Sulforaphane suppresses the growth of triple-negative breast cancer stem-like cells in vitro and in vivo

doi: 10.1158/1940-6207.CAPR-18-0241

Figure Lengend Snippet: ELISA assessment of CR1 binding to solid phase BPs including Nodal, GRP78, and Alk4 in the absence (-S) or presence (+S) of 100µM of SFN. CR1 binding to individual BPs in the absence of SFN (-S) condition was normalized to 100%. Percentage reaction induced CR1 binding inhibition was then determined using the 100% reference point for respective BPs. Data represents average values determined from three separate studies with samples run in octuplicate.

Article Snippet: CR1 (R&D Systems, Cat# 145-CR/CF), Nodal (R&D Systems, Cat# 3218-ND-024/CF), GRP78 (Abcam, Cat# ab78432), and Alk4 (Creative BioMart, Cat# ACVR1B-645H,) were purchased from the indicated vendors.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition