GAB1 levels and AKT phosphorylation is increased during ibrutinib therapy. (A) Representative immunoblot of MEC1 cells treated with ibrutinib (2 µM) or vehicle (dimethyl sulfoxide [DMSO]) for the indicated time points. Ibrutinib and DMSO were freshly supplemented every 24 hours, and the cells were sampled at the indicated time points for immunoblot analysis. (B-C) FoxO1 and GAB1 mRNA expression in MEC1 cells treated with ibrutinib or DMSO, as described in panel A (n = 5). (Di) Representative immunoblot for primary CLL cells isolated from peripheral blood of patient before (Pre) and during ibrutinib therapy. For the patients’ characteristics, see supplemental Table 1. *β-Actin was the endogenous control for p-p85 and t-p85, assayed on a separate gel with identical loading. (Dii) Statistical analysis of normalized densitometric data from immunoblots of CLL samples (n = 11) obtained from patients before (Pre) and during ibrutinib therapy. The data are relative to expression before ibrutinib administration (set at 1). For additional immunoblots, see supplemental Figure 23. (E) GAB1 and FoxO1 mRNA expression in primary samples isolated from patients with CLL before (Pre) and during ibrutinib therapy (n = 11; for patients’ characteristics, see supplemental Table 1). The data are relative to expression before ibrutinib (set as 1). (F) Representative immunoblot of MEC1^WT and MEC1^GAB1-KO cells treated with ibrutinib (1 µM) or vehicle (DMSO) for 7 days. *Decrease in total BTK protein level is a common effect of ibrutinib, as described previously.⁵⁸