Pig CD47 does not interact with mouse SIRPα. (A) Western blot analysis of SIRPα tyrosine phosphorylation in WT mouse macrophages. Macrophages were incubated in medium alone (control; lane 1), or with CD47^−/− mouse (lane 2), WT mouse (lane 3), or porcine (lane 4) RBCs for 30 minutes. (Rows 1-2) Macrophage lysates were used directly in Western blot with anti–β-actin (row 1, as a loading control) or with antiphosphotyrosine Ab (α-pTyr; row 2). (Row 3) Macrophage lysates were immunoprecipitated by anti-SIRPα mAb P84; precipitated proteins were then analyzed by Western blot with antiphosphotyrosine Ab (α-pTyr). A representative experiment of 3 is shown. (B) Blocking SIRPα by anti-SIRPα mAb (P84) augments phagocytosis of WT mouse, but not CD47^−/− mouse or porcine RBCs. CFSE (green)–labeled splenic macrophages (5 × 10⁵/well) were incubated with or without anti-SIRPα antibody (P84) in 96-well plate for 20 minutes; then PKH-26 (red)–stained WT mouse (WT), CD47 KO mouse (CD47^−/−), untreated pig (pRBC), or opsonized pig (ops pRBC) RBCs (1 × 10⁶/well) were added and phagocytosis was determined 1 hour after incubation using fluorescent microscope (engulfment was seen as a yellow event). The percent of macrophages engulfing target cells per well was calculated as follows: [number of yellow events/(number of yellow events + number of green nonengulfing macrophages)] × 100%. Data are presented as mean ± SDs (n = 10-12 wells per group). **P < .01.