Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

doi: 10.4049/jimmunol.1202451

Figure Lengend Snippet: FIGURE 1. Characterization of the recom- binant CR1 CCP 22–30_His fragment. (A) SDS-PAGE analysis of 4 mg CR1 CCP22-30 under reducing (R) and nonreducing (NR) conditions. The positions of the m.w. mark- ers are indicated. (B) CD spectroscopy of CR1 CCP22–30. A spectrum was recorded in the far-UV (200–260 nm) and collected six times. The mean values for each wavelength were calculated. The maximal ellipticity is indicated by an arrowhead. (C) Electron microscopy analysis of CR1 CCP22–30 (375000) after negative staining with 2% sodium silicotungstate.

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

Techniques: SDS Page, Circular Dichroism, Electron Microscopy, Negative Staining

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The production yield, activity, and purification of recombinant Cripto produced in the 3D microcarriers as compared to the 2D method. ( A ) SDS-PAGE analysis of the purification steps of recombinant Cripto: lane M is the protein molecular weight marker; lane 1 is the protein solution from ultrafiltration; lane 2 is the flowthrough after the first passage through the His-tag affinity Ni-NTA resin; lane 3 is the flowthrough after the second passage through the same resin; lane 4 is the first wash step; lane 5 is the second wash step; lane 6 is the first elution from the His-tag affinity Ni-NTA resin; and lane 7 is the second elution from the same resin. The band at approximately 27 kDa is the Cripto protein (indicated by the red arrow). ( B ) Quantitative amounts of Cripto protein produced in the 3D batch (Cripto (3D) ) with HEK293 cells encapsulated in PF microcarriers and incubated in bioreactors after three rounds of harvesting are compared to the maximum amount of Cripto produced in the 2D batch (Cripto (2D) ) with HEK293 cells adherent to cell culture plates and cultured to their density threshold limits. An initial cell seeding of 3.2 × 10 6 cells was used for both techniques. ( C ) The biological activity of recombinant Cripto was compared for Cripto produced in PF microcarriers versus the 2D method by measuring binding affinity to the AlK4 receptor. Four independent experiments were carried out for the 3D system and three independent experiments were performed for the 2D cultivation method. Results are shown as mean ± S.D. *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Activity Assay, Purification, Recombinant, Produced, SDS Page, Molecular Weight, Marker, Incubation, Cell Culture, Binding Assay

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The effect of Cripto produced in 3D microcarriers on C2C12 cell proliferation detected by the BrdU incorporation assay. ( A ) C2C12 myoblast proliferation was evaluated by the BrdU incorporation assay after being cultured for 48 h in serum-free medium containing Cripto produced in 3D microcarriers (Cripto (3D) ) or commercially available Cripto (Cripto (R&D) ). Also evaluated were a bFGF medium positive control and a serum-free medium negative control. ( B ) The recombinant Cripto induces myoblast proliferation in a dose-dependent pattern; increasing concentrations of Cripto (3D) and Cripto (R&D) were added to C2C12 cells, and proliferation was quantified by the BrdU incorporation assay. ( C ) Cell proliferation was further evaluated by counting the total number of live cells. The proliferative effect of Cripto (3D) was compared with that of commercial Cripto (R&D) . The data are presented as mean ± S.D. from at least three independent experiments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Produced, BrdU Incorporation Assay, Cell Culture, Positive Control, Negative Control, Recombinant

Journal: Journal of Biological Chemistry

Article Title: Growth Factor Induction of Cripto-1 Shedding by Glycosylphosphatidylinositol-Phospholipase D and Enhancement of Endothelial Cell Migration

doi: 10.1074/jbc.m702713200

Figure Lengend Snippet: FIGURE 1. Lipid raft localization and release of CR-1 protein. A, NTERA2/D1 cells were stained with anti-CR-1 mAb (green), and lipid raft marker GM-1 was labeled with CTxB (red) without permeabilization and analyzed by confocal microscopy (upper panel). Magnified images for the marked regions in the upper panel are shown in the lower left panel. Negative controls without pri- mary antibody are shown in the lower right panel. Nuclei were stained with DAPI (blue) in all merged images. Scale bar 10 m. B, biochemical isolation of DRMs with 1% Triton X-100 by sucrose-gradient centrifugation of NTERA2/D1 cells. The gradient fractions (lanes 4–12) were analyzed with Western blot analysis for CR-1. Transferrin receptor (TfR) was used as a control for non-raft membrane protein. The lipid raft marker GM-1 in the same sam- ples was detected with HRP-conjugated CTxB by dot-blot analysis. C, compar- ative study of cell-associated (CL) and released (CM) CR-1 with or without serum by semi-quantitative Western blot analysis (see Table 1 and supple- mental Fig. S1, A–C). A representative blot for NTERA2/D1 cells is shown. The indicated amount of recombinant human CR-1 protein was used as a standard.

Article Snippet: Reagents—Human CR-1 monoclonal antibodies (mAbs) (MAB2771 and FAB2772P) were obtained from R&D Systems (Minneapolis, MN) or developed as previously reported (B3F6) (8).

Techniques: Staining, Marker, Labeling, Confocal Microscopy, Isolation, Gradient Centrifugation, Western Blot, Control, Membrane, Dot Blot, Recombinant