Generation of mouse CD47-expressing rat cell line.

<p>(A) Structure of pRc/CMV-mouse CD47. The entire coding region of the mouse CD47 cDNA was PCR-amplified. The amplified PCR product was digested and full-length mouse CD47 cDNA was inserted into the expression vector pRc/CMV. (B) Expression of mouse CD47 on a transfected rat insulinoma cell (INS-1E) was confirmed by FACS analysis. Representative histograms obtained by FACS analysis for mouse PBMCs, pRc/CMV-transfected rat INS-1E cells (cont-INS-1E), and pRc/CMV-mouse CD47-transfected rat INS-1E cells (mCD47-INS-1E) are shown. Open and filled histograms represent staining with isotype control and with anti-mouse CD47 mAb, respectively. (C) Tyrosine phosphorylation of SIRPα in mouse macrophages was induced by incubation with pRc/CMV-mouse CD47-transfected rat INS-1E cells (mCD47-INS-1E), but not with control vector-transfected rat INS-1E cells (cont-INS-1E). Differentiated mouse macrophages were incubated with mCD47-INS-1E or cont-INS-1E at 37°C for 30 min. The cells were lysed, and the lysates were mixed with mouse anti-mouse SIRPα antibodies and 50% slurry of protein G-sepharose beads by rotation at 4°C for 8 hrs. Precipitated proteins were separated by 8% SDS-PAGE, followed by blotting to a nitrocellulose membrane. Rabbit immunoaffinity-purified anti-phosphotyrosine IgG and goat anti-rabbit HRP-conjugated IgG were used as primary and secondary antibodies, respectively. Mouse CD47-transfected INS-1E (mCD47-INS-1E) alone (lane 2), mouse macrophages incubated in medium alone (lane 3) or mouse macrophages incubated with cont-INS-1E (lane 4) or mCD47-INS-1E (lane 5) are shown. Immunoblotting with anti-mouse SIRPα was used as loading control. IP, immunoprecipitation; IB, immunoblotting; anti-pY, anti-phosphotyrosine.