Effects of Y78F and Y112F mutations on signaling pathways activated by TpoR. Ba/F3 cells retrovirally transduced with Δ5TpoR or TpoRW515A and mutants thereof (Y78F or Y112F) were sorted for similar expression levels of GFP and examined for protein expression (A), activation of JAK2 (B), and activation of several downstream signaling pathways (C). Detection was performed by Western blotting with anti–phospho-specific antibodies that recognize the major phosphorylation sites that are linked to activation of catalysis (for kinases and phosphatases) or transcription (for STAT proteins). JAK2 activation was assessed by Western blotting with anti–phospho-JAK2 Y1007, as phosphorylation of activation loop Y1007 is obligatory for activation of JAK2 signaling. The Y78F mutation enhanced JAK2 activation and signaling via TpoRwt and Δ5TpoR. The Y112F mutation did not alter JAK2 activation, but inhibited downstream signaling, especially by Δ5TpoR.