Differential activation of AKT pathway by ECD and PTD mutants. (A) Constitutive association of p85 with all KIT mutants. Cells were serum starved for 5 hours and then stimulated with or without mSCF for 5 minutes. KIT proteins were immunoprecipitated, fractionated by SDS-PAGE, and blotted with anti-P85 (top panel) or anti-KIT antibodies (bottom panel). (B) Activation of AKT and its downstream pathway by ECD and PTD mutants. Total lysates from serum-starved Ba/F3 cells were subjected to SDS-PAGE and immunoblotted with antiphospho-AKT (T308), antiphospho-AKT (S473), and antiphospho-GSK3β (S9) antibodies. Membranes were stripped and reprobed with anti-AKT antibody. The experiment was performed 3 times with similar results.