Expression of IL-11 receptor α chain in human BM CD41⁺CD14⁻ cells. (A) CD34⁺ human BM cells were isolated and cultured as described in the Materials and Methods. Total RNA from sorted cultured CD41⁺CD14⁻ cells and K562 and HEL cells (positive controls for IL-11 receptor α chain and c-mpl mRNAs, respectively) was isolated, first-strand cDNA was synthesized, and gene expression was analyzed via RT-PCR. The fragments were visualized by staining with ethidium bromide after separation in 2% agarose gel. The primers used generated the following fragments: human IL-11R, 475 bp (lanes 1, 4, and 6); human c-mpl, 379 bp (lanes 3 and 5); and human GAPDH, 361 bp (lane 2). Lane 1, no cDNA; lane 2, MK cDNA from 2.5 × 10⁴ cell equivalents; lanes 3 and 4, MK cDNA from 10⁵ cell equivalents; lane 5, HEL cDNA from 37.5 ng of polyA⁺ RNA; lane 6, K562 cDNA from 37.5 ng of polyA⁺ RNA. (B) Western blot analysis was performed on total cell lysate from human cultured BM CD41⁺CD14⁻ cells and K562 cells (positive control). CHO/IL11R (James Tobin, Genetics Institute, Inc) cells were used as a positive control for IL-11 receptor α chain antibody specificity and CHO cells as the negative control. Total cell protein from CD41⁺CD14⁻ cells (3 × 10⁶ cells for IL-11R and 5 × 10⁵ cells for CD41) and from cell lines (2 × 10⁶ K562 cells for IL-11R, 5 × 10³ CHO/IL-11R cells for IL-11R, and 5 × 10³ CHO cells for IL-11R) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. IL-11 receptor α chain, 48-kD protein (lanes 1 through 4), and CD41, 135-kD protein (lane 5), were detected with the appropriate antibodies (1.0 μg/mL). Autoradiography was performed on the membranes (CD41 for 4 minutes and IL-11 receptor for 1 hour).