Signal transduction characteristics of cEPOR mutants in BaF3 cells. (A) Whole cell lysates of the BaF3 transfectants described in Fig 1 were subjected to immunoprecipitation with anti-JAK2 antiserum followed by separation by polyacrylamide gel electrophoresis and immunoblotting analysis with anti-phosphotyrosine antibody. Cells were either resting (0) or stimulated with EPO at low-dose (1, 1 U/mL) or high-dose (10, 10 U/mL) EPO for 15 minutes before lysis. The blots were reprobed with anti-JAK2 antibody to verify the identity of the phosphoprotein band and equivalent gel loading (not shown). (B) Nuclear extracts prepared from resting or EPO-stimulated BaF3 cell lines were prepared and subjected to EMSA with an oligonucleotide probe containing a STAT-binding sequence (FcγRI). Supershift analysis with anti-STAT5 antisera was used to confirm the identity of the STAT within the retarded nucleoprotein complex (not shown). (C) Whole cell extracts prepared from resting (0) or EPO-stimulated (1 U/mL or 25 U/mL, as indicated) BaF3 cell lines were prepared and subjected to in vitro kinase assays as described in Materials and Methods.