Cyclin D3 gene expression is upregulated by Mpl ligand in Y10 cells, a subclone of the mouse megakaryocytic cell line L8057, at the (A) mRNA level, (B) protein level, and (C) transcriptional level. Y10 cells were cultured in the presence or absence of MGDF, as detailed in Materials and Methods. (A) Northern blot analysis of 15 μg total RNA, using cDNAs of cyclin D3 and cyclin A as probes. Equal loading of RNA per lane was confirmed by probing the blot with cDNA encoding 18S rRNA. (B) Western blot analysis of 10 μg protein from whole cell lysates of Y10 cells, using a cyclin D3 antibody. (C) Nuclear run-on assays, performed as described in Materials and Methods. Ten micrograms of plasmid containing cDNA for cyclin D3, 2 μg of plasmid containing cDNA for 18 S rRNA, or 10 μg pUC 19 was applied to nitrocellulose. The intensities of the bands were quantitated using the Electrophoresis Documentation and Analysis System. The data presented are of a representative experiment out of three performed.