Ras expressions and protein activities. (A) Quantitative reverse-transcribed PCR was performed to determine Nras and Kras mRNA levels in normal hematopoietic cells that were sorted from bone marrow of 3-month-old WT F1 mice. Different populations were defined according to Figure 3A. The level of GAPDH was used as a loading control. The relative levels of Nras or Kras to GAPDH were determined in 4 independently sorted bone marrow samples. Expression in whole marrow (WBM) was normalized to a value of 1 for comparison with subpopulations. (B) Western blot of subpopulations from 3-month-old F1 bone marrow probed with N-Ras and K-Ras specific antibodies. Actin level was used as a loading control. (C) Activated, GTP-bound Ras proteins were immunoprecipitated from WT, Kras mutant, and Nras mutant cells and probed with pan Ras antibody (top). Total protein lysates were probed with pan Ras (middle) and actin (bottom) antibodies. (D) Quantitative reverse-transcribed PCR of Nras and Kras mRNAs from WT, Kras mutant, and Nras mutant bone marrow cells.