Determination of cell-cycle regulatory protein status by Western blot in CD34⁺ hematopoietic progenitors transduced with mutant Ras. CD34⁺ control, CD34⁺ N-Ras^G12D, or CD34⁺ H-Ras^G12V cells were incubated for 16 hours in serum-replete, growth factor–free medium in the presence or absence of 1μM DPI. Expression and phosphorylation state of key cell-cycle regulatory proteins in whole-cell lysates were determined by Western blot. In all cases, GAPDH was used as a loading control. (A) The endogenous expression of cell-cycle promoters cyclin D3 and cyclin D1 and cell-cycle inhibitors p15^INK4B and p21^Cip1 was determined. (B) Cytosol/nuclear fractionated lysates were probed with antibody recognizing the cell-cycle inhibitor p21^Cip1. (C) Whole-cell lysates derived from both CD34⁺ control and CD34⁺ H-Ras^G12V cells (treated with either DPI or vehicle control) were probed with antibodies recognizing the cell-cycle promoters CDK6, CDK4, cyclin D1, and cyclin D3. (D) Whole-cell lysates as in panel C were probed with antibodies recognizing the following cell-cycle inhibitory proteins; p21^Cip1 (which inhibits CDK/cyclin complexes); p15^INK4B (which sequesters CDK4/6); p27^Kip1 (which has a similar role to p21^Cip1) and p38^MAPK, both total and phosphorylated (T180/T182).