Rho activity can be stimulated by TPO during MK differentiation. CD41⁺ MKs derived from leukapheresis were cytokine-starved overnight in serum-free medium and then stimulated for 5 minutes and 120 minutes with 100 ng/mL TPO. Pull-down RBD-bound Rho and total Rho in the lysate were detected by Western blot. (A) Cultured MKs starved on day 6 and stimulated on day 7 (left panel) or starved on day 11 and stimulated on day 12 (right panel). (B) Western blot quantification showing the Rho-GTP/total Rho ratio at day 7 and day 12 of culture. Results are the average ± SD of 3 experiments. The same number of cells were used for pull-down analysis at day 7 and day 12 of culture. In this experiment, the total quantities of Rho augmented from day 7 to day 12 because the MK size markedly increases in culture during this time lapse. Thus, the ratio between Rho-GTP and total Rho permits evaluation of the relative activity of Rho. Quantification results show that TPO-stimulated Rho activity measured at 5 minutes decreased of about 22% at day 12 compared to day 7 (t test; P < .01).