Signaling through CRLF2 F232C requires JAK2 but not phosphorylation of CRLF2 Y368. (A) siRNA against mouse Jak2 was transfected into Ba/F3 cells expressing the indicated alleles and proliferation was assayed after 48 hours (*P < .05, **P < .01 compared with control siRNA in the same line by paired, Student t test). (B) Western blotting for indicated proteins after siRNA transfection. (C) Ba/F3 cells expressing the indicated alleles were grown for 48 hours in the absence of cytokines and in the presence of increasing concentrations of compounds. RFU indicates relative fluorescence units. All assays were performed in quadruplicate. Error bars indicate the SEM (***P < .001 compared with Ba/F3-CRLF2 F232C/JAK2). (D) IP of HA-tagged CRLF2 constructs from Ba/F3 cells expressing the indicated alleles followed by WB for CRLF2 phospho-Y368 or HA. Where indicated, cells were pretreated for 15 minutes with pervanadate. FC indicates F232C. (E) IP using anti-CRLF2 antibody in Ba/F3 cells expressing WT CRLF2 or CRLF2 Y368F (YF) with WT IL7R or IL7R-3F (3F) followed by immunoblotting with a specific antibody against CRLF2 P-Y368. IGH indicates mouse immunoglobulin heavy chain. (F) MHH-CALL4 (M4) and MUTZ-5 (M5) human B-ALL cells and Ba/F3-CRLF2/JAK2 R683G cells were treated with vehicle or pervanadate and then lysates were collected and immunoblotted for the indicated proteins. (G) Ba/F3 cells expressing the indicated combinations of CRLF2 and IL7R alleles were cultured in the absence of IL3 and cells were counted every other day. Error bars indicate SD.