Nuclear and cytoplasmic localization of BTK, ERK, and AKT in persistent lymphocytes following ibrutinib therapy and ability of persistent lymphocytes to stimulate. Nuclear and cytoplasmic localization of BCR signaling pathway proteins was evaluated using nuclear and cytoplasmic lysates from patients at baseline and after 6 months of ibrutinib therapy. In these representative immunoblots, there is no evidence of a shift toward nuclear localization of either phosphorylated or total (A) BTK, (B) ERK, or (C) AKT. (D) Patient samples at baseline and after 9 months of therapy were used to evaluate whether persistent lymphocytes can be stimulated through the BCR (15-minute exposure to plate-immobilized IgM; BD Pharmigen), TLR9 (3.2 μM CpG; Eurofins MWG Operon), or distal to BTK (1 μg/mL CD40L; PeproTech). (D) In this representative immunoblot, mild stimulation with CpG is seen at baseline along with robust stimulation following CD40L. After 9 months of ibrutinib therapy, CD40L alone is able to stimulate CLL cells.