PlGF is produced by erythroid cells and is expressed in SCD bone marrow. (A) RNA was extracted from normal peripheral blood mononuclear cells (composed mainly of monocytes and lymphocytes, lanes 1 and 2) and an erythroid culture derived from normal bone marrow (lane 4) and subjected to RT-PCR analyses. PlGF primers used spanned 2 exons, giving a larger intron-containing band for genomic DNA (lane 3). (B) RT-PCR analysis on normal bone marrow cells (lanes 1 and 2), SCD bone marrow (SBM, lane 3), β-thalassemia major bone marrow (TBM, lane 4), and 2 normal umbilical cord blood mononuclear cells (UCB, lanes 5 and 6) using PlGF (top panel) and β-actin (bottom panel) primers. Lanes 7-10 are control lanes: H₂0 (lane 7) and the Molt T-cell line (lane 8) as negative controls, 293 cell genomic DNA as a DNA control (lane 9), and endothelial cells (lane 10) as a positive control. (C) RT-PCR analyses for PlGF, glycophorin A (an erythroid cell–specific gene), and β-actin followed by transfer and probing, showing the proportion of PlGF transcription in normal versus SCD light density mononuclear cells. The short (PlGF^S) and long (PlGF^L) exposure of the PlGF blot is shown to depict the small amount of PlGF mRNA in normal bone marrow and relatively high amount in sickle bone marrow. (D) Western blot analyses of glycophorin A⁺ (Gly⁺) and glycophorin A^– (Gly^–) light density mononuclear cells (LD-MNCs). Lanes 1 and 2 represent Gly⁺ and Gly^– cells from SCD LD-MNCs (SBM), respectively; lanes 3 and 4 represent Gly⁺ and Gly^– cells from normal bone marrow LD-MNCs (NBM), respectively; lanes 5 and 6 represent fibroblasts and placenta, as negative and positive controls, respectively. (E) RT-PCR analysis for PlGF on the NBM Gly⁺ and Gly^– cells, shown in panel D (lanes 1 and 2). Lanes 3 and 4 represent controls.