JAK2 mutant Ba/F3-MPL cell line generation. (A) Ba/F3 cell lines that stably express Mpl (BaF-MPL) and 1 of 4 types of human JAK2 (WTJAK2, JAK2R564Q, JAK2V617F, and both JAK2R564Q and JAK2V617F) were generated. (B) No significant difference (n.s.d) is seen in levels of human/mouse JAK2 cDNA levels between the 4 cell lines, measured by real-time PCR. (C) Western blot analysis of total JAK2 and total MPL levels in each of the 4 mutant cell lines compared with Ba/F3-MPL parental cells (first lane from left). (D-E) Cell surface expression of MPL in the Ba/F3-MPL-JAK2 cell lines, analyzed by flow cytometry. (D) Examples of overlays of the surface MPL flow cytometry of stained cells (black peak) compared with BaF3 parental controls (unfilled peak) for all 4 cell lines. (E) Histogram representing the combined flow cytometric data from all 3 repeats. No significant difference (n.s.d.) in surface MPL expression was found between the 4 cell lines.
