Expression of Pim-1 constructs in stably transfected BaF3 cells. (A) Expression of Pim-1 constructs was detected by monoclonal anti-Pim-1 antibody from abstracts of transfected cell lines. Extracts (100 μg/sample) were resolved by separation in 12% polyacrylamide gels and immunoblotted with anti-Pim-1 antibody. Lane 1 shows BaF3 cells deprived of IL-3 for 8 hours; lane 2, BaF3/ITD; lane 3, Pim-1/44 (44 kDa); lane 4, Pim-1/33 (33 kDa); lane 5, Pim-1/NT81 (27.5 kDa). (B) Expression of Pim-1, FLT3, and Actin after treatment with or without CEP-701 for 1 hour. Pim-1 and Actin were directly assessed by immunoblotting with the anti-Pim-1 and anti-Actin antibodies. FLT/ITD expression and phosphorylation were detected by immunoprecipitation with FLT3 antibody (S-18) followed by immunoblotting with 4G10.