FIGURE 1.

Activation of JAK2-V617F signaling and transforming properties by a heterodimeric receptor component, IL27Ra. A, an empty expression vector (lanes 1 and 2), and expression vectors for IL27Ra (lanes 3 and 4) or EpoR (lanes 5 and 6) were transfected into 293T cells along with a wild type JAK2 (WT) expression vector (lanes 1, 3, and 5) or a JAK2-V617F (VF) expression vector (lanes 2, 4, and 6). Two days after transfection, cells were lysed and an equal amount of lysate protein was analyzed by immunoblotting using antibodies to detect pJAK2 (Tyr¹⁰⁰⁷/Tyr¹⁰⁰⁸), pSTAT5 (Tyr⁶⁹⁴), pERK (Thr²⁰²/Tyr²⁰⁴), JAK2, STAT5, ERK, IL27Ra, and EpoR (via HA), as indicated. B, BaF3 cells co-expressing empty vector (lanes 1 and 2), IL27Ra (lanes 3 and 4), or EpoR (lanes 5 and 6) with wild type JAK2 (WT) (lanes 1, 3, and 5) or JAK2-V617F (VF) (lanes 2, 4, and 6) were blotted with antibodies to detect JAK2 and STAT5 (as a loading control). Lysate of BaF3 cells expressing an empty vector control was included (lane 8) to ascertain the approximate level of exogenous JAK2 expression in BaF3 cells expressing either wild type JAK2 or JAK2-V617F in lanes 1–6. C, cytokine-dependent BaF3 cells co-expressing an empty vector (lanes 1 and 2), IL27Ra (lanes 3 and 4), or EpoR (lanes 5 and 6) with wild type JAK2 (WT) (lanes 1, 3, and 5) or JAK2-V617F (VF) (lanes 2, 4, and 6) were starved of cytokine for 3 h. Cells were then lysed and an equal amount of lysate protein was analyzed by immunoblotting for pJAK2 (Tyr¹⁰⁰⁷/Tyr¹⁰⁰⁸), pSTAT5 (Tyr⁶⁹⁴), pAKT (Ser⁴⁷³), JAK2, STAT5, and AKT. Lysate from cytokine-independent cells expressing IL27Ra with JAK2-V617F and EpoR with JAK2-V617F are shown in lanes 8 and 9, respectively. In the lower panel, similar lysates are immunoblotted for pERK (Thr²⁰²/Tyr²⁰⁴) and ERK, with the lysate from cytokine-independent IL27Ra/JAK2-V617F cells shown in lane 6. D, BaF3 cells co-expressing control vector (left), IL27Ra (center), or EpoR (right) with wild type JAK2 (circles) or JAK2-V617F (squares) were washed and plated in the absence of cytokine on day 0. Total viable cells were determined by trypan blue exclusion over time. The broken line indicates the viable cell count dropped below the limit of detection of the hemocytometer.