MPLS204P is a weak gain-of-function mutant. (A) Signaling studies induced by high TPO concentration in MPLWT and MPLS204P UT7 cells. After cytokine deprivation, cells were stimulated with TPO (100 ng/mL for 5 minutes, 30 minutes, or 2 hours). The phosphorylation status of STAT3, STAT5, AKT, and ERK1/2 was examined by western blotting and results were compared with cells stimulated by GM-CSF (5 ng/mL). (B) STAT5 transcriptional activity in γ2A JAK2-deficient cells expressing MPLWT or MPLS204P. Cells were transfected with cDNAs coding for MPLWT or MPLS204P, JAK2, STAT5, and with Firefly STAT5 luciferase reporter spi-Luc and pRL-TK vectors coding for renilla luciferase. Luminescence was measured after 48 hours’ transfection. Shown are average units ± SEM of 1 representative experiment performed in triplicate of 3. (C) Persistent signaling in UT7 MPLS204P. UT7 MPLWT and UT7 MPLS204P were stimulated by TPO (10 ng/mL) for 10 minutes and subsequently washed. A 3-hour time course was performed followed with western blotting, as in previous figures. MPLWT and MPLS204P samples were run on the same gel. P-STAT5/STAT5 or P-ERK/ERK ratios are indicated after quantification by image J software.