Expression of Nox mRNA in MKs. (A top) RT-PCR of Nox1 and Nox4 in magnetic bead–purified Wt MKs. RNA was prepared from purified MKs and BM fractions from cultured BM (+TPO; see purification image in Figure 2). Mouse VSMC cDNA was used as a positive control for detection of Nox1 (262 base pairs [bp]) and Nox4 (315 bp) transcripts. Omitting reverse transcriptase (RT) was used as a control for the PCR, whereas amplification of GAPDH was used as a control for RNA preparation (554 bp transcript). (A bottom) Determination of the purity of the MK-enriched fraction. RNA was prepared as described above. Amplifications of the neutrophil marker Ly6G and of macrophage/monocyte marker CD68 were used to exclude the possibility of contamination from different BM cell populations. Mac indicates macrophages. (B) Detection of Nox1 mRNA in the murine megakaryocytic Y10 cell line. RT-PCR was performed on Y10 cDNA from nontreated and TPO-treated (for 24 hours) cultures. VSMC cDNA was used as a positive control for detection of Nox1 and Nox4 transcripts. (C) Effect of TPO on Nox1 mRNA levels. Quantitative RT-PCR analysis was applied in RNA isolated form purified MKs after vehicle or TPO stimulation of the BM culture. 18s rRNA was used as an internal control, and Nox1 levels were normalized to CD41 expression levels (a MK marker). The graph represents the average of 3 independent experiments. Statistical significance (*P < .05) was determined using the Student t test.