CRLF2 box1, W286R and Y368F mutations in CRLF2/mutant JAK2 signaling. (A-D) Ba/F3 cells expressing the indicated alleles were grown in the absence of cytokines beginning day 0 and cells were counted every other day. Insets show immunoblotting for the indicated proteins. (E) Immunoblotting was performed with antibodies against indicated total or phospho (P−) proteins either in the presence of IL3 or 24 hours after IL3 withdrawal. Tyrosine phospho-sites are included. (F) Coimmunoprecipitation of FLAG-tagged JAK2 with CRLF2 constructs in Ba/F3 lysates. (G) Coimmunoprecipitation of GST or GST-CRLF2int (WT or carrying the indicated mutations) generated in E coli with HA-tagged JAK2 generated by in vitro translation. P/S indicates box1-P/S; WR, W286R; YF, Y368F; RG, R683G; and VF, V617F.