Expression of C3aR by human CD34+ cells and C3 in bone marrow extracts.
(A) RT-PCR analysis of C3aR expression in normal human BM- (lane 1), PB- (lane 3), and CB-derived (lane 5) CD34+ cells. Lanes 2, 4, and 6 show negative control (H2O instead of mRNA) RT-PCR reactions. (B) FACS analysis of C3aR expression on human CD34+ cells sorted by magnetic beads. Data presented are from a representative experiment, which was repeated 3 times with similar results. We found that 34 ± 11% of CD34+ cells express C3aR. (C) Calcium flux studies of Fura-2–loaded normal mobilized PB CD34+ cells. C3a (1 μg/mL) was added at the indicated time points, and calcium flux was evaluated by spectrophotofluorimetry. Data are from a representative experiment, which was repeated 3 times with similar results. (D) Western blot analysis of expression of C3 and its cleaveage products in bone marrow extracts from 4 different mice. The α- and β-chains of murine C3 are shown in lane 1 (muC3). BM1 to BM4 analysis of C3 and its cleavage products (68-kDa and 45-kDa fragments of α-chain) in murine bone marrow extracts. The presence of a 68-kDa fragment suggests generation of C3a.
