Functional analysis of JAK2 mutants. (A) STAT-dependent transcriptional activity induced by wild-type JAK2 or mutants of JAK2. γ2A cells which are JAK2-deficient were transiently transfected with wild-type JAK2 (or wild-type JAK2 and mutant JAK2 at a 1:1 ratio to represent heterozygous condition), MPL, STAT5, and with Firefly STAT5 luciferase reporter spi-Luc and pRL-TK vector coding for Renilla luciferase. Luminescence was measured 24 hours after transfection. After transfection, medium was changed after 5 hours and replaced either with culture medium or medium supplemented with TPO (10 ng/mL). Shown are means ± SEM of 3 independent experiments done in triplicate. **P < .001; *P < .05. (B-C) The viability of Ba/F3 cells expressing wild-type MPL together with wild-type JAK2 or different mutants was assessed after 72 hours in the presence of increasing concentrations of IL-3 (B) and TPO (C). Error bars represent SEM. (D) The activation of STAT5 in starving condition. Ba/F3 cells expressing the wild-type MPL only, or together with wild-type JAK2 or JAK2 mutants were cultured for 48 hours on TPO (1 ng/mL) and then starved for 4 hours in serum-free medium without TPO. Western blot was performed on the cell lysates with antibodies against pYSTAT5, STAT5, hemagglutinin-tag (HA), and JAK2. An antibody against Hsc70 was used as loading control.