CD28 signaling induced PI3K activation. The indicated MM cells were treated with 1 μg/mL of agonistic anti-CD28 mAb 9.3 for different time course and fixed. Levels of phosphorylated PI3K were assayed by FACE PI3 Kinase p85 ELISA kit (Active Motif) per the manufacturer's instructions. Mean + SEM is shown. Data are performed in triplicate for each time point and is representative of 2 independent experiments. The x-axis (time) is nonlinear. Compared with time 0, at 5 minutes P = .11 for 8226, P = .006 for U266, and P = .02 for MM1.S. Compared with time 0, at 1440 minutes P = .13 for 8226, P = .017 for U266, and P = .005 for MM1.S.