Selective inhibition of JAK2^V617F protein expression and signaling by mt1- and mt4-siRNAs. (A) Sequences of the siRNAs designed to specifically knock down the JAK2^V617F, but not the JAK2^WT protein production. Mutated nucleotides are indicated in bold, and their positions are designated from the 5′ end of the Jak2^V617F homologous sequence. HEL and UT-7 cells were transfected (siRNA) or not (nontreated) with the indicated siRNAs. HEL cells only express JAK2^V617F, whereas UT-7 cells only express JAK2^WT. (B) The JAK2 and β-actin (loading control) proteins were analyzed in UT-7 and HEL cells by Western blotting with the specific antibodies, 48 hours after treatment. Results show that the mt1- and mt4-siRNAs, but not the mt2- and mt3-siRNAs, were efficient in specifically knocking down JAK2^V617F (only present in HEL), but not JAK2^WT (only present in UT-7). The Jak2-siRNA (JAK2), used as a positive control, knocked down both JAK2^WT and JAK2^V617F. Western blotting also assessed the phosphorylation levels of the JAK2 downstream effectors STAT5 and ERK1/2 in HEL cells. Jak2-, mt1-, and mt4-siRNAs all caused a marked decrease in STAT5 and ERK1/2 phosphorylation without affecting their total protein level. Neither mt2- nor mt3-siRNA had an effect on JAK2 downstream signaling.