recombinant human cr1  (GenScript corporation)

Journal: International Journal of Nanomedicine

Article Title: An Immunoreceptor-Targeting Strategy with Minimalistic C3b Peptide Fusion Enhances SARS-CoV-2 RBD mRNA Vaccine Immunogenicity

doi: 10.2147/IJN.S463546

Figure Lengend Snippet: Confirmation of targeting proficiency for mC3 and mFc ligands via immunofluorescence assay (IFA). ( A ) Illustration of the ligands-receptors interactions that were demonstrated by IFA. Following mRNA transfection into 293T cells, receptors were incubated with the cells. Then, RBD-mC3 bound CR1 while RBD-mFc bound FcγR. The RBD-mC3 and RBD-mFc were labeled by anti-RBD monoclonal antibodies. The CR1 and FcγR were marked using anti-CR1 polyclonal antibodies and anti-FcγR monoclonal antibodies, respectively. ( B ) Antibodies targeting CR1 (FITC, green) and RBD-mC3 (Cy5, purple) depicted the respective distributions of CR1 and RBD-mC3. ( C ) Colocalization analysis of RBD-mC3 and CR1. ( D ) Antibodies against FcγR (green) and RBD-mFc (purple) showed the distributions of FcγR and RBD-mFc respectively. The blue color (DAPI) represented the cell nuclei. ( E ) Colocalization analysis of RBD-mFc and FcγR. Three regions of interest (ROI) were analyzed. Pearson’s coefficient (R) was calculated in colocalization analysis. R > 0.8 suggests a very strong correlation.

Article Snippet: Then, the cells were blocked with 10% goat serum (Jackson ImmunoResearch) for 1 hr at room temperature, followed by one-hour incubation of receptor proteins, including recombinant human CD35 protein (CR1) (R&D Systems #5748-CD-050, NE Minneapolis, MN) and mouse CD64/FCGR1 protein (Sino Biological #50086-M08H).

Techniques: Immunofluorescence, Transfection, Incubation, Labeling

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: Amino acid (AA) sequence comparison of human CR1 (Accession No. AAH67844.1) and human CR3 (Accession No. AAG49539.1). The “*” indicates AA differences between CR1 and CR3. Yellow highlight = signal peptide. Fuchsia highlight = Abcam anti-CR1 MoAb ab108391 binding site (Leu44-Arg66). Red highlight = EFG-like domain (Nodal binding). Blue highlight = Abcam anti-CR1 MoAb ab133236 binding site (Cys97-Glu113). Purple highlight = CFC domain (GRP78/Alk4 binding). Green highlight = Glycoslylphosphatidylinositol (GPI) linkage domain. Double-headed arrow indicates the site of GPI-phospholipase D cleavage, resulting in the formation of soluble protein products. CR3A = peptide immunogen (srgdlafrdds) conjugated to KLH and used to generate anti-CR3 mouse MoAbs. CR3B = negative control peptide (srgylafrdds). CR1A = peptide immunogen (qrvppmgiqhs) conjugated to KLH and used to generate anti-CR1 mouse MoAbs. CR1B = negative control peptide (qrvlpmgiqhs).

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Sequencing, Comparison, Binding Assay, Negative Control

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: GenScript Biotech Corporation mouse anti-CR1 and anti-CR3 generation data.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Clone Assay

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: GenScript Biotech Corporation mouse MoAb production data and resulting titration curves for NCI final candidate choice. ( A ) Titration curve for anti-CR1 MoAb 5G1-1 evaluated on the following, solid-phased: Blue—R&D Systems recombinant human CR1, fuchsia—CR1A peptide immunogen, green—CR1B negative control peptide, gray—R&D Systems recombinant mouse CR1, yellow—R&D Systems recombinant human cryptic, orange—MyBioSource recombinant human CR3, black—CR3A peptide immunogen, and red—CR3B negative control peptide. ( B ) Titration curve for anti-CR3 5G11-2 evaluated on the following, solid-phased: Orange—MyBioSource recombinant human CR3, black—CR3A peptide immunogen, red—CR3B negative control peptide, blue—R&D Systems recombinant human CR1, fuchsia—CR1A peptide immunogen, green—CR1B negative control peptide, gray—R&D Systems recombinant mouse CR1, and yellow—R&D Systems recombinant human cryptic.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Titration, Recombinant, Negative Control

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: Biacore/surface plasmon resonance analysis of anti-CR1 NCI 5G1-1 MoAb and anti-CR3 NCI 5G11-2 MoAb binding to recombinant human CR1 or CR3. ( A ) NCI 5G1-1 MoAb (blue) diluted at 0.8, 4, 20, 100, and 500 nM versus NCI 5G11-2 (orange) at the same test concentrations measured interaction with immobilized CR1. ( B ) NCI 5G11-2 MoAb (orange) diluted at 0.8, 4, 20, 100, and 500 nM versus NCI 5G1-1 (blue) at the same test concentrations measured interaction with immobilized CR3.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: SPR Assay, Binding Assay, Recombinant

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: Dilution of test antibodies giving 50% deflection of binding titration curve via solid-phase ELISA.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: IHC staining of human normal/tumor tissue. ( A ) Lung IHC analysis: ( A1 ) Normal lung, airway epithelium −CR3/+CR3, ( A2 ) Squamous CA, +CR1/+CR3, ( A3 ) Adenosquamous CA, tumor vascular endothelium +CR1/−CR3 (Anatomical Separation), tumor cells −CR1/+CR3, ( A3′ ) Enlargement of +CR1 blood vessels—black arrowheads, ( A4 ) Adenocarcinoma, +CR1/+CR3, ( A5 ) Adenocarcinoma, tumor vascular endothelium +CR1/−CR3 (Anatomical Separation), and tumor cells −CR1/+CR3, and ( A5′ ) Enlargement of +CR1 blood vessels—black arrowheads. ( B ) Colon IHC analysis: ( B1 ) Normal colon, cryptic epithelium +CR1/+CR3, ( B2 ) Adenocarcinoma, tumor vascular endothelium +CR1/−CR3 (Anatomical Separation Variant), tumor cells +CR1/+CR3, ( B2′ ) enlargement of +CR1 blood vessels—black arrowheads, ( B3 ) Adenocarcinoma, tumor cells −CR1/+CR3, ( B4 ) Adenocarcinoma, tumor vascular endothelium +CR1/−CR3 (Anatomical Separation Variant), tumor cells +CR1/+CR3, ( B4′ ) enlargement of +CR1 blood vessels—black arrowheads, and ( B5 ) Adenocarcinoma, tumor cells −CR1/+CR3. ( C ) Breast IHC analysis: ( C1 ) Normal breast, lobular ductal epithelium +CR1/+CR3, ( C2 – C5 ) Invasive ductal adenocarcinoma, tumor cells +CR1/+CR3. ( D ) Ovary IHC analysis: ( D1 ) Normal ovary mesothelium +CR1/+CR3, ( D2 ) Normal pellucida of the primary follicles +CR1/+CR3, ( D3 ) Normal cells of the inner theca +CR1/+CR3, ( D4 ) Endometrioid adenocarcinoma −CR1/+CR3, ( D5 ) Metastatic adenocarcinoma +CR1/+CR3, ( D6 ) Dysgerminoma −CR1/+CR3, and ( D7 ) High-grade serous carcinoma +CR1/+CR3. ( E ) Prostate IHC analysis: ( E1 ) Normal prostate −CR1/−CR3, ( E2 ) Normal-looking tissue adjacent to adenocarcinoma +CR1/+CR3, possibly representing a premalignant lesion, ( E3 ) Adenocarcinoma +/−CR1/+CR3, ( E4 ) Adenocarcinoma +CR1/+CR3, and ( E5 ) Adenocarcinoma −CR1/+CR3. Photographs taken at 20× magnification and size bar constant for all figures.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Immunohistochemistry, Variant Assay

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: IHC detection of CR1/CR3 in human tissue and ranking of TMN_N, TMN_M, grade, stage, and progesterone receptor expression based on staining intensity. ( A ) Discriminate TMN_N0 from TMN_N1 in prostate cancer as scored by CR1 staining intensity. ( B ) Ranking of TMN_M0 vs. 5TMN_M1 in prostate cancer as revealed by CR1 staining. ( C ) Stage values for prostate cancer tracks with CR1 staining. ( D ) Discriminate colon mucinous adenocarcinoma grades +, ++, & +++ by CR3 staining. ( E ) Ranking of colon adenocarcinoma grades +, ++, and +++ based on CR3 staining. ( F ) Progesterone receptor score tracks with CR3 staining in breast cancer. Photographs taken at 20× magnification, and size bar constant for all figures. Dots indicate outliers.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: CR1 versus CR3 expression in human tumor cell lines as revealed by Western blot (WB) analysis. Note: CR1 and CR3 band intensity values normalized to respective GAPDH levels for the analyzed sample. Identification of human tumor cell lines and negative control. MCF7—breast CA, MDA-MB231—triple-negative breast CA, HepG2—hepatocellular CA, HT29—colorectal adeno CA, A549—bronchioloalveolar CA, hmVECs—immortalized endothelial cells. ( A ) Combined WB data for CR1 versus CR3 and corresponding housekeeping GAPDH loading standard. ( B ) Quantitative analysis of relative CR1 versus CR3 expression levels in cell lysates normalized to GAPDH loading control. Uncropped blots are shown in .

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Expressing, Western Blot, Negative Control, Control

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: CR1/CR3 capture/quantitative ELISA for evaluation of soluble CR1/CR3 proteins in sera. ( A ) Representative CR1 capture/quantitative ELISA standard curve along with respective inserted linear equation graph used to determine CR1 values [ng/50 mL/well] of unknown serum sample. ( B ) Representative CR3 capture/quantitative ELISA standard curve along with respective inserted linear equation graph used to determine CR3 values [ng/50 mL/well] of unknown serum sample. ( C ) CR1 levels [ng/mL] identified in normal female donors and breast cancer patient sera. ( D ) CR3 levels [ng/mL] identified in normal female donors and breast cancer patient sera.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Human Cripto-1 and Cripto-3 Protein Expression in Normal and Malignant Settings That Conflicts with Established Conventions

doi: 10.3390/cancers16213577

Figure Lengend Snippet: CR1/CR3 interaction with solid-phased established CR1-binding proteins (Nodal, GRP78, and Alk4). To validate our assay was working correctly, we utilized positive control solid-phased antibodies that recognized antigenic epitopes on CR1/CR3 distal from NCI MoAb binding sites. These included the Abcam anti-CR1 N-terminal target (ab108391) = aNTCR1 and the anti-CR1 EGF-like domain target (ab133236) = aEGFCR1. The interaction of CR1 or CR3 to solid-phased Nodal, GRP78, or Alk4 proteins was detected with, respectively, anti-CR1 NCI 5G1-1 or anti-CR3 NCI 5G11-2 MoAbs. ( A ) CR1 interaction with solid-phased binding proteins or controls in the presence or absence of equimolar CR3. ( B ) CR3 interaction with solid-phased binding proteins or controls in the presence or absence of equimolar CR1. ( C ) CR1 percent binding inhibition with equimolar CR3. ( D ) CR3 percent binding inhibition with equimolar CR1. All target proteins were solid-phased at [50 ng/50 μL/well] overnight and blocked with HFBTS.

Article Snippet: Recombinant human CR1 (GenScript Biotech, Piscataway, NJ, USA) and CR3 (MyBioSource, San Diego, CA, USA) standard curves were made via serial dilutions starting with 50 ng/50 μL/well followed by 2-fold dilutions to 0.0244 ng/50 μL/well.

Techniques: Binding Assay, Positive Control, Inhibition

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: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Electrophoretic mobility (Mr) and gene frequency of the allelic size isoforms of CR1.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques:

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Characteristics for Analyzed Plasma and Reb Blood Cell CR1 Samples.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques:

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: CR1 immunofluorescence staining using mouse mAbs 8C9.1(A), E11 (B) or J3B11 (C) on fixed CHO cells expressing 1x10 6 (high) (upper panels) or 1x10 5 (moderate) (middle panels) receptors/cell. Bottom panels show isotype control on high CHO-CR1 (A), or antibody reactivity on non-transfected CHO cells (B, C). Scale bar: 50 μm.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Immunofluorescence, Staining, Expressing, Transfection

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Representative micrographs of brain sections stained with monoclonal anti-CR1 Abs 8C9.1 (A, B, D-F), J3B11 (G-I) or mIgG control (C) in frontal cortex of control (CTR) and Alzheimer’s disease (AD) cases. Genotype for the two GWAS SNPs rs4844609 (T or A) and rs6656401 (G or A) are presented in each panel. Scale bar 100 μm.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Staining

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Immunostaining with monoclonal anti-CR1 8C9.1 (A, B) or J3B11 (C) without (upper panels) or with (lower panels) preabsorption with recombinant human CR1. Neuronal staining (B) was not preabsorbed with rhCR1. Scale bar: A, C: 50 μm, B: 100 μm.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Immunostaining, Recombinant, Staining

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Immunofluorescent dual labelling of CR1 (8C9.1, red) and astrocytes (anti-GFAP, green) in an AD case (A-F). Merged images show colocalization (C) or lack of colocalization (F) of CR1 and GFAP immunofluorescence labelling. Scale bar: 50 μm.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Immunofluorescence

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: (A) WB of RBC samples with and without SNP rs6656401 (loaded 0.9 ng CR1 per lane) using E11 mAb. Lanes: 1 RBC control, 2 C (nondemented) (AT/AA), 3 MCI (mild cognitive impairment) (AT/AG), 4 C (AT/AG), 5 MCI (AT/AG), 6 C (TT/GG), 7 MCI (TT/GG), 8 C (TT/AG), 9 MCI (TT/GG). (B) WB of brain extracts from donors with and without rs6656401 (loaded 100 μg brain extract protein per lane 3–9) probed with E11 mAb. Lane: 1 sCR1 (1 ng), 2 RBC (5 μg), 3 C (TT/AG), 4 AD (AT/AA), 5 AD (AT), 6 AD (TT/AA), 7 AD (TT/GG), 8 C (TT/GG), 9 C (AT/AG). Exposure time in lanes 1 and 2 was 5 sec and in lanes 3–9, 7 min. (C) Immunoprecipitates of E11 (4 μg) (lanes 3, 5, 7) or IgG1 (4 μg) (lanes 2, 4, 6) with sCR1 (2 μg) (lanes 2,3), RBC from a MCI subject (TT/GG) (50 μg protein, lane 4, 5) or brain extracts from cognitively intact subject (AT/AG) (250 μg, lane 6, 7). sCR1 is loaded at 1 ng (lane 1). (D) Representative immunoprecipitations of brain lysates C (TT/GG) (250 μg protein) (lanes 3–6) with 8C9.1 (lane 4), J3B11 (lane 6), or corresponding isotype IgG (lanes 3, 5). Control sCR1 loaded at 1 ng (lane 1) and RBC extract loaded at 1 ng CR1 (lane 2). C and D. Blots were probed with polyclonal anti-CR1 Ab.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques:

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: Soluble CR1 was measured by ELISA and plotted vs. A. SNP [rs6656401 only (n = 21) or rs6656401 and rs4844609 (n = 16) and without the rs6656401 and rs4844609 SNPs (n = 22)] or B. categorized by diagnosis [nondemented (n = 22),), MCI (n = 14) and AD (n = 19)]. Recombinant CR1 was used to generate the standard curve. Black symbols indicate samples with no variant SNP (rs6656401 and rs4844609), red reflects donors with two SNPs, and blue is donors with rs6656401 only. Data points are from multiple experiments in which samples were run in triplicate and normalized to standard CR1 samples across experiments.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Variant Assay

Journal: PLoS ONE

Article Title: Analysis of the Putative Role of CR1 in Alzheimer’s Disease: Genetic Association, Expression and Function

doi: 10.1371/journal.pone.0149792

Figure Lengend Snippet: CR1 partially purified from RBC was assessed for binding to immobilized C1q (A, B) or C3b (C, D, E, F). A. UCI cohort: CR1- C1q relative binding for CR1with rs6656401 (n = 10) only, with rs6656401 and rs4844609 (n = 8), or without rs6656401 or rs4844609 (n = 11) SNPs. Black, green and purple symbols indicate patients clinically diagnosed as cognitively normal, mild cognitive impairment (MCI), and demented (AD) respectively. B. UCI cohort: CR1-C1q relative binding vs diagnosis: Normal (n = 12), MCI (n = 11) and Dementia (n = 16). In C and E, CR1-C3b relative binding is plotted by CR1 isoform (determined by WB): donors with common allele (fast form or F form) (n = 12) or with extra C3b-binding domain (n = 17) (slow form or S form) in UCI cohort (C) or F form (n = 38), S form (n = 17) for SRI cohort (E). In D and F, CR1-C3b relative binding plotted vs diagnosis: Normal (n = 12), MCI (n = 11), Dementia (n = 6) in UCI cohort (D) or normal (n = 30), MCI (n = 13), Dementia (n = 20) in SRI cohort (F). (Reference CR1 samples were used to standardize values among assays performed on different dates). In B-F: samples with no variant SNP (black); red, donors with two SNPs (rs6656401 or rs4844609); blue, donors with rs6656401 only and green open square is donor with unknown SNP genotype. Subjects in whom the CR1 SNP analysis was discordant with the F/F WB results are visible as blue circles in Fig 9C and E. *p<0.01–0.05, **p<0.01–0.001, calculated using 1 way ANOVA. Data points are from multiple experiments in which samples were run in triplicate and normalized to standard CR1 samples across experiments.

Article Snippet: In addition, anti-CR1 mAbs 8C9.1 and J3B11 were preabsorbed with recombinant human CR1 (R&D, Minneapolis, MN) (1:15 molar ratio) before incubation with the tissue to validate specificity of staining.

Techniques: Purification, Binding Assay, Variant Assay