Figure 2.

T/P transforms Ba/F3 cells to IL-3 factor independence. (a) Ba/F3 cells were transfected with pcDNA3, pcDNA3 T/P, the kinase-inactive mutant, Y635K, or the ΔHLH mutant and selected for G418 resistance. G418-resistant cells were selected for growth in the absence of IL-3. Neo-resistant (2 × 10⁴ cells; pcDNA3, Y635K, and ΔHLH) or IL-3-independent cells (T/P) were washed free of IL-3 and plated on day 0 in RPMI 1640/10% FCS. Viable cells were counted on each day. (b) Expression of T/P and mutants in Ba/F3 cells. Transfected Ba/F3 cells were lysed in 1% Triton X-100/150 mM NaCl/50 mM Tris, pH 8.0, plus protease inhibitors. Lysates were separated by SDS/PAGE, transferred to nitrocellulose, and blotted with anti-PDGFβR antisera. Lanes 2 and 3 show two separate transfections with wild-type T/P.