CRLF2 box1, W286R and Y368F mutations in TSLP and CRLF2 F232C signaling. (A) Ba/F3 cells expressing the indicated alleles were grown in TSLP beginning day 0 and cells were counted every other day. Inset shows immunoblotting for the indicated proteins. (B) Immunoblotting was performed with antibodies against indicated total or phospho (P−) proteins. Tyrosine phospho-sites are included. Abbreviations: WT, wild-type; P/S, box1-P/S; WR, W286R; and YF, Y368F. (C) Identical to panel A except cells were grown in the absence of cytokines. (D) Immunoblotting as in (B) P-STAT5/STAT5 indicates the ratio of band intensities quantified for each lane. (E) Ba/F3 cells expressing WT CRLF2 or CRLF2 F232C alone or with HA-tagged JAK2 R683G were subjected to immunoprecipitation (IP) using an antibody against total JAK2 followed by immunoblotting (Western blot [WB]) for the indicated proteins. WCL indicates whole cell lysates.