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Blood, Vol. 107, Issue 6, 2548-2556, March 15, 2006
Species- and cell type-specific interactions between CD47 and human SIRP
Blood Subramanian et al.
107: 2548
Supplemental materials for: Subramanian et al
SUPPLEMENTAL MATERIALS AND METHODS Flow cytometry
For flow cytometry, forward scatter, side scatter and fluorescence (FL1, FL2, FL3 channels in logarithmic mode) were acquired for at least 10,000 events using a FACScan or FACSCalibur (BD Immunocytometry Systems, San Jose, CA). Microscopy
Images were acquired on a Nikon TE300 inverted microscope with a 4× or 60× (oil, 1.4 NA) objective using a Cascade CCD camera (Photometrics, Tuscon, AZ). Image acquisition was performed with Image Pro software (Media Cybernetics, Silver Spring, MD). All image analysis was done using Image J 1.31v (http://rsb.info.nih.gov/ij). ELISA
Immulon 2HB 96-well plates (Thermo Electron, Waltham, MA) were incubated with 100 µl protein solution in PBS for at least 1hr at room temperature. Wells were incubated with 200 µl blocking solution (PBS containing 1% BSA and 0.05% Tween 20) for at least 1 hr at room temperature. Primary antibody in 100 µl volume was added to wells and incubated for at least 1 hr at room temperature. The antibody solution was removed and wells were washed 3× with 200 µl blocking solution. AP- or HRP- conjugated secondary antibody was added in a 100 µl volume and incubated for at least 30 min at RT. Antibody was removed and wells were washed with blocking solution. Before addition of substrate, wells were washed with PBS or 100 mM Tris-HCl. 200 µl of substrate (pNPP for AP or ABTS for HRP; Sigma-Aldrich) was added and absorbance at 405 nm was monitored for 5-10 min. The slope in the linear portion of the absorbance time profile was used for quantification. To determine absolute values for protein density, saturating levels of fluorescently labeled protein was added to wells and total and bound fluorescence was measured using a fluorimeter (Fluoroskan Ascent FL, Thermo Electron). The absolute amount of bound protein could thus be obtained since the solution concentration (measured using theoretical extinction coefficient and absorbance measurements at 280 nm) was known. This method could not be used when lower concentration of proteins was used, and the ELISA was used to determine the bound protein levels. AFM Probing of RBC
Human and rat packed RBC were diluted and mixed together and 100 µl of this suspension was allowed to adhere to a poly-L-lysine coated (10 mg/ml in coating solution) glass slide for 10 min. Unattached cells were removed by gentle rinsing of the slide several times with PBS solution, and an additional volume of PBS was added for the experiments. Blunt tips (Microlevers, Park Cantilevers) were first silanized by immersion in a solution of 1.25% ATC in toluene. The silanized tips are then immersed in recombinant human SIRP ex solution at high concentration (0.5 mg/ml) or low concentration (0.05 mg/ml) for 10 minutes for functionalization. The functionalized tips were then washed thoroughly in 1% BSA solution in PBS to remove loosely attached protein. Force curves in vertical indentation and/or retraction of spread erythrocytes were obtained with an Asylum Research AFM (at ∼5 µm/sec) mounted on a Nikon Eclipse TE 300 inverted microscope. Western Blotting
Cell lysates were prepared from frozen cell pellets lysed on ice in PBS containing 1% Triton-X-100 and protease inhibitors (AEBSF, E-64, bestatin, leupeptin, aprotinin, & Na-EDTA). Cell debris was then removed by centrifugation. Purified protein and cell lysates were denatured for 10 min at 100°C with SDS and  -mercaptoethanol (Fisher Scientific, Hampton, NH) and then left untreated or incubated with PNGase (New England Biolabs, Beverly MA) or Endo H (New England Biolabs) at 37°C overnight. Samples were then electrophoresed on Bis-Tris gels (NuPAGE; Invitrogen) under reducing conditions in MOPS buffer and transferred to 0.2 µm PVDF (BIORAD, Hercules, CA). The membrane was blocked with 5% non-fat milk in TTBS (Tris-buffered saline with 1% Tween 20) by shaking for 1 hour at room temperature or overnight at 4ºC, washed with TTBS and incubated with primary antibody in TTBS for at least 1 hour with gentle shaking. After repeated washing, the membrane was incubated with alkaline phosphatase conjugated goat secondary antibody (Sigma-Aldrich) in TTBS for at least 30 minutes. The membrane was washed in TTBS and then in TBS and incubated with the alkaline phosphatase substrate, NBT-BCIP.
Files in this Data Supplement:
- Figure S1. Species-restricted binding of CD47-antibodies to RBC (PDF, 29.6 KB) -
RBC from five mammalian species were incubated with human CD47 antibodies. Bound protein was detected using fluorescent secondary antibodies and flow cytometry under standardized conditions of cell number and reagent concentration. Blocking and non-blocking antibodies show distinct, species-restricted binding profiles. The cross-reactivity of human CD47-specific antibodies to CD47 on RBC from multiple species (Table 2) was studied because a subset of these antibodies blocked SIRP. It was therefore expected that the patterns obtained from the entire set would provide insight into the location of the SIRP binding site. Only BRIC126 showed a pattern of binding that closely matches human SIRP1ex binding data. Another SIRP-blocking antibody, B6H12 did not show any reactivity to pig CD47. The reactivity of 6H9 and C1km1 indicated that pig, human and cow CD47 could be distinguished as a group from rodent CD47. mIAP301, an antibody against mouse CD47, did not bind human CD47 confirming that there are differences in the epitopes in or near the SIRP binding domain of human and murine CD47. The non-blocking antibody 2D3, bound only human CD47 and lead to enhanced human SIRP1ex binding (Figure 2A), suggesting that this antibody recognizes a unique epitope removed from the SIRP binding domain.
- Figure S2. Mouse SIRPαex binding to RBC is also species-specific (PDF, 33.5 KB) -
Human RBC or mouse RBC were incubated with GST, soluble human SIRP1ex or mouse SIRPex and bound protein was detected in flow cytometry using fluorescent anti-GST. Mouse SIRPex binding to mouse RBC is CD47-specific since mIAP301 antibody reduces binding to background levels (data not shown).
- Figure S3. Species-specific binding of soluble glycosylation variants of human SIRPα1ex to RBC (PDF, 35.8 KB) -
RBC from five mammalian species were incubated with aglycosylated SIRP1-GST. Bound protein was detected using fluorescent secondary antibodies and flow cytometry under standardized conditions of cell number and reagent concentration. Binding pattern is identical that seen in Figure 2D.
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