Evaluation of hRasGRP4 expression in peripheral blood. A, Evaluation of hRasGRP4 mRNA levels in unfractionated peripheral blood mononuclear cells (PBMCs), PBMC-derived CD14+ cells, CD14-/CD3-/CD19- cells and T cells isolated from healthy individuals. A RT-PCR/gel separation approach using an exon 1/exon 17 primer set in the hRasGRP4 gene was carried out to evaluate transcript expression in each cell type. Human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH)-specific primers were used in the lower panels as positive controls. Representative results from three independent experiments are shown. B, A qPCR approach using a primer set that recognizes the junctional part of exon 7 and exon 8. Delta-CT of hRasGRP4 transcript level relative to the hGAPDH transcript in CD14+ cells was defined as 1. qPCR assay was done in a triplicate manner for three times and the error bars indicate standard errors. C, Immunohistochemistry; PBMC-derived CD14+ myeloid cells and T cells were stained with anti-hRasGRP4 antibody (top panels). For negative and positive controls, HEK293 cells that differed in their levels of hRasGRP4 protein also were stained with the anti-hRasGRP4 antibodies. For additional controls, replicate cells were stained with anti-β-actin antibodies (middle panels) or irrelevant rabbit IgG (bottom panels). Representative results from three to four procedures are shown.