To assess whether CD28 binding to its ligands CD80 and CD86 triggers PI3K signaling, we cultured U266 and RPMI 8226 myeloma cells (which constitutively express CD28, Supplemental Fig. 1) with anti-CD28-coated beads (to mimic stimulation by ligands expressed on antigen-presenting cells), in the absence or presence of a CD28-blocking mAb, and used Western blotting to examine the expression of components of the PI3K/AKT signaling pathway (Fig. 1A, B). Here, AKT1 is linked to plasma cell myeloma.