Functional analysis of SEC31A-JAK2. (A) IL3 deprivation of Ba/F3 cells transduced with SEC31A-JAK2 and with the deletion mutant ΔWD40-JAK2 resulted in transformation to growth factor–independent growth. When transduced with the deletion mutants WD40-JAK2, PRO-JAK2, and ΔJAK2, Ba/F3 cells were not able to proliferate in the absence of IL3. The mean growth ± SEM of 3 separate measurements over 5 consecutive days is shown. (B) Schematic representation of the protein structures of the SEC31A-JAK2 deletion mutants. (C) SEC31A-JAK2, SEC31A-ALK, and JAK2^V617F–expressing Ba/F3 cells, cultured in the absence of IL3, were treated with the indicated concentrations of the JAK inhibitors CP690550 (left panel) and JAK inhibitor I (right panel), and cell survival was quantified after 24 (SEC31A-JAK2 and SEC31A-ALK) or 48 (JAK2^V617F) hours. Ba/F3 cells expressing SEC31A-JAK2 and JAK2^V617F, but not SEC31A-ALK, were inhibited in a dose-dependent manner by treatment with JAK inhibitors. IC₅₀ values for CP690550 were observed between 500nM and 1μM for SEC31A-JAK2 and between 100nM and 500nM for JAK2^V617F. Treatment with JAK inhibitor I resulted in IC₅₀ values between 100nM and 500nM for SEC31A-JAK2 and between 10nM and 50nM for JAK2^V617F. Cell survival in the absence of inhibitor was set at 100%. Mean ± SEM of 4 independent measurements is shown. (D) Western blot analysis showing the effect of CP690550 (left panel) and JAK inhibitor I (right panel) treatment on Ba/F3 cells transduced with SEC31A-JAK2, grown in the absence of IL3. In addition to SEC31A-JAK2 phosphorylation (158 kDa), phosphorylation of AKT, STAT3, and STAT5 decreased with increasing CP690550 concentration. In cells treated with JAK inhibitor I, phosphorylation of MAPK (ERK1 + ERK2), STAT3, and STAT5 reduced with increasing inhibitor concentrations. Expression of total SEC31A-JAK2 (158 kDa), MAPK (ERK1 + ERK2), AKT, STAT3, and STAT5 remained unaffected.