Modulation of downstream pathways and dimerization of full length hTpoR by JAK2 WT and JAK2 V617F. (A) Representative western blotting of phosphorylation pattern of major signaling pathways from Ba/F3 stably expressing indicated constructs. (B) Left: STAT3 phosphorylation in Ba/F3 cells expressing either hJAK2 WT or hJAK2 V617F together with indicated constructs. Values represent mean fluorescence intensity (MFI) of 3 to 4 independent experiments. Right: Representative flow cytometry measurement of STAT3 phosphorylation in Ba/F3 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (C) Left: STAT1 phosphorylation in Ba/F3 cells expressing either hJAK2 WT or hJAK2 V617F together with the indicated constructs. Values represent the mean fluorescence intensity (MFI) of 4 to 5 independent experiments. Right: Representative flow cytometry measurement of STAT1 phosphorylation in Ba/F3 cells expressing hJAK2 WT (blue) or hJAK2 V617F (red) and the indicated constructs. (D) Illustration of the live-cell crosslinking assay. Dimerization of human TpoR was assessed by introducing cysteine point mutations in the singleton of human TpoR, which contains the entire extracellular domain. Pretreatment with N-ethylmaleimide (NEM) prevents nonspecific crosslinking of extracellular cysteines and all intracellular cysteines are removed by truncation of the intracellular domain after the JAK2 binding domain. The membrane-permeable cysteine crosslinker o-phenylenediamine (o-PDM) has a 4 Ä arm-length and allows the stable crosslinking of cysteines at the interface. (E) Representative western blot in denaturing and reducing conditions of monomers (bottom) and crosslinked dimers (top) of hTpoR WT and cysteine mutants in the presence of JAK2 WT with and without Tpo, or JAK2 V617F with or without Tpo. (F) Helical wheel diagram illustrating the position of each amino acid mutated to cysteines 1 by 1. Interfaces represented by each of the 7 cc-hTpoR are shown with roman numbers from 0 to VI.