Regulation of CD150-mediated Akt activation. (A) Cross-linking of CD150 on WT CD150⁺ DT40 transfectants did not induce Akt phosphorylation on S473. Lyn and Btk were involved in negative regulation of CD150-mediated Akt pathway. SHP-2^-/- DT40 transfectant showed constitutive high level of pAkt phosphorylation, but neither BCR nor CD150 ligation on Syk-deficient cells was able to trigger the Akt pathway. (B) Cotransfection of CD150 and SH2D1A resulted in rapid CD150-mediated activation of Akt that was SHIP-independent. Wild-type DT40 transfected with empty pApuro or pApuro/pcDNA3 vectors served as negative controls. Western blot analysis on cell lysates. Each sample contained lysate from 10⁶ cells. Equal loading was monitored by anti-p38 MAPK kinase or anti-actin Western blot in all experiments (control panels). One of 5 experiments.