Phosphorylation of PLCɣ and ERK1/2 induced by bead-immobilized anti-IgM in CLL SGs. Phosphorylation of PLCγ2 (pPLCγ2, A) or of ERK1/2 (pERK1/2, B) induced after exposure to bead-bound anti-IgM was measured in SGs 1 to 4 in the FACS Calibur. Percent of phosphorylated cells was calculated as the differential between the percent of cells phosphorylated at 37°C (activated) and that at ice temperature (background). (C) A representative case (CLL9) showing inhibition of pERK1/2 by ibrutinib in each individual SG. Filled histogram represents ERK1/2 phosphorylation in CLL cells incubated on ice, dashed line ERK1/2 phosphorylation at 37°C in the absence of ibrutinb, and continuous line ERK1/2 phosphorylation at 37°C in the presence of ibrutinib. (D) Effect of ibrutinib on ERK1/2 phosphorylation in SG4. Mean and SEM of the 6 cases analyzed are represented (CLL3, 7, 8, 9, 11, and 12).