Expression of CDCP1 in bone marrow stromal cells.

<p><b>Panel A</b>: Gene expression of CDCP1 in HS27a, HS5 and 2 primary stromal cultures was determined by qPCR. Y-axis shows relative gene expression of CDCP1. <b>Panel B</b>: Western blot (left panel) of Triton-X-100 extracts of HS27a and HS5 cells before (−) and after (+) P3D9 stimulation. Sixty micrograms of cellular proteins were loaded in each lane. Antibodies against CDCP1 were used for Western blotting. M.W. stands for molecular weight markers. Right panel shows protein staining with Coomassie Brilliant Blue to show the equal protein load. <b>Panel C</b>: Immunofluorescence staining of CDCP1 in HS27a cells (upper panel) and HS5 cells (lower panel). Scale bars, 20 µm. <b>Panel D</b>: Protein expression of CDCP1 on the surface of HS27a (black) and HS5 (blue) cells was determined by flow cytometry using P1C3 antibody against CDCP1. Flow cytometry using additional monoclonal anti-CDCP1 antibodies (CUB1, P5H10 and P3D9) which recognize different epitopes on CDCP1 showed the same results (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109304#pone.0109304.s001" target="_blank">Figure S1 in File S1</a>). Gray area within black line is the isotype-matched control. <b>Panel E</b>: Proportion of CDCP1+ and CD146+ stromal cells in primary marrow cultures. Bone marrow mononuclear cells were isolated from five healthy donors and primary long-term stromal cultures were established. The cells were stained with antibodies against CD146, CDCP1, CD14 and CD45, and were analyzed by flow cytometry. The proportion of CDCP1+ (left panel) and CD146+ (right panel) stromal cells are shown on the y-axis.