Reduction of Ptpn2 expression levels accelerates the transformation process of activating JAK1 mutants. (A) Transformation assay of Ba/F3 cells expressing various mutant forms of JAK1. Expression of 4 different JAK1 mutants conferred cytokine-independent growth on Ba/F3 cells. Simultaneous reduction of Ptpn2 expression (shRNA-Ptpn2-A; black bars) enhanced the transformation process compared with control cells (shRNA-control1; white bars). The y-axis displays the number of viable cells. Cell numbers ± SEM were recorded in triplicate. Data shown are representative of 3 independent experiments. *P < .05. (B) Western blot analysis of cytokine-independent Ba/F3 cells. shRNA-mediated knockdown of Ptpn2 resulted in increased phosphorylation of JAK1 and Stat5 compared with control cells (shRNA-control1). Five different JAK1 mutants are shown. Efficient knockdown was confirmed using a Ptpn2 antibody. β-actin was used as an independent loading control.