Effects of TPO, activin A, BMP2, and BMP4 on markers of late stages of MK maturation. CD34⁺ cells were incubated for 3 to 18 days in 4GF serum-free medium in the presence or not of 50 ng/mL TPO, activin A (ACT), BMP2, or BMP4 as indicated. (A) After 3 days, we assayed the expression of PF4 and FOG-2 at the RNA level by RT-PCR analysis, as described in “RNA isolation and analysis.” In all conditions, we found a similar level of expression of the housekeeping gene GAPDH, which allowed us to compare levels of expression of PF4 and FOG-2 between the different treatments. (B) After 7 days of culture, total cell lysates were quantified, and 10 μg total proteins was separated by SDS-PAGE. Immunodetection was performed as described in “Western blot analysis” using monoclonal mouse anti-PF4 and anti-CD42a antibodies, or antiactin used as a loading control. (C) Finally, at the latest stages of in vitro MK differentiation and using 30 μL cell culture supernatant, secreted PF4 was detected after 10 and 18 days of culture. Equal aliquots of the culture supernatant were separated by SDS-PAGE. Immunodetection was performed as described in “Western blot analysis” using monoclonal mouse anti-PF4 antibody.