MTT and Western blotting analyses. (A) (Left) MTT assay of mutant-type c-Mpl. W1 and W2 are the transfectants with the wild-type allele (Ser505), and M1 and M2 are those with the mutant type (Asn505). The x-axis indicates time of culture after withdrawal of IL-3. The y-axis shows the relative value of cell viability (OD, 580-630 nm). Half the culture medium was exchanged with IL-3-free medium every other day. Mutant cells (Asn505) exhibited survival capacity in the absence of IL-3 (each experiment was repeated 3 times). (Right) Western blot with the c-Mpl antibody. Arrow shows the c-Mpl products (82 kDa). The FDCP2 cells transfected with the wild-type c-MPL were used as a positive control. The lysate of Ba/F3 cells and the plasmid PCI-neo carrying no foreign gene were analyzed as negative controls. The same filter was stripped and blotted with β-actin antibody. (B) Western blot with the Mek1/2 antibody of Ba/F3 transfectants and immunoprecipitation-Western blot with p-Tyr and Stat5b antibodies. Phosphorylated Mek1/2 signals (45 kDa) were detected in M1 and M2 after 6 hours culture with the IL-3-free medium. The same filter was stripped and blotted with anti-Mek1/2 antibody. The phosphorylated Stat5b signals of 96-kDa products were observed in M1 and M2 in the IL-3-free condition. The same filters were stripped and blotted with anti-Stat5b antibody. This experiment was repeated twice and the results were reproducible. (C) Western blot with the Mek1/2 antibody of the platelets from the ET-affected member. The constitutively phosphorylated Mek1/2 was detected in the platelets of the ET-affected individual (patient `a' in Figure 1A) even in the absence of the stimulation of TPO.