TPO concentrations in MF and normal plasmas, as well as MPL expression by MF and normal CD34⁺ cells. (A) TPO levels in MF and normal plasmas were measured using an enzyme-linked immunosorbent assay as described in supplemental Methods. The TPO concentration was significantly increased in MF plasma as compared with normal plasma. N plasma: n = 6; MF plasma: n = 13. (B) The expression of MPL by primary normal BM and MF CD34⁺ cells was determined by western blotting with a polyclonal Ab raised against the C-terminal amino acids of MPL. (C) Densitometric analysis of western blots as represented by (B) shows that the total level of MPL protein was reduced by 34% in MF CD34⁺ cells as compared with normal BM CD34⁺ cells. MF: n = 9; N BM: n = 6. (D-F) Expression of MPL on primary normal BM and MF CD34⁺ cells was evaluated by flow cytometry using a mAb, which specifically identifies the extracellular domain of MPL. (D) Representative flow cytometric plot showing MPL expression by normal BM (N BM14), MF splenic (SP19), and PB (PB195) CD34⁺ cells. (E) MPL expression was significantly reduced on MF splenic and PB CD34⁺ cells and CD34⁺Lin⁻ CD38⁻ cells, as compared with normal BM counterparts. (F) Moreover, JAK2V617F⁺ and JAK2V617F⁻ MF CD34⁺ cells and CD34⁺Lin⁻CD38⁻ cells showed a similar degree of reduction of MPL expression. *P < .05; **P < .01; ***P < .001. N BM: n = 8; MF splenic: n = 11; MF PB: n = 7; JAK2V617F⁺ MF: n = 9; and JAK2V617F⁻ MF: n = 9. N BM, normal BM; N plasma, normal plasma; SP, splenic.