Vemurafenib, dabrafenib, and trametinib cause sustained, dose-dependent MEK and ERK dephosphorylation in primary HCL but not HCL-like cells. (A-B, D) Western blot analysis of purified HCL cells from 4 representative patients shows strong phosphorylation of both MEK and ERK under basal conditions (0 nM drug, ie, DMSO vehicle only) and their dose-dependent dephosphorylation after 30 minutes to 72 hours of incubation with the specific active BRAF inhibitors vemurafenib and dabrafenib (A-B) at concentrations ranging from 100 nM to 1000 nM, and with the MEK inhibitor trametinib (D) at concentrations ranging from 10 nM to 100 nM. (C) Conversely, primary leukemic cells from a representative HCL-like patient express a relatively low basal level of phospho-ERK which is not influenced by treatment with up to 1000 nM vemurafenib for 30 minutes to 6 hours. Membranes were probed with antibodies against phospho-ERK1/2 (pERK), phospho-MEK1/2 (pMEK), total ERK1/2, and total MEK1/2 as indicated on the left of each panel. Please note that to obtain, from the HCL-like cell lysates, the pERK bands representatively shown in panel C, the exposure of the pERK blot had to be prolonged (tens of minutes) as compared with HCL cell lysates (tens of seconds in A-B). Solid and dashed lines separate lanes repositioned from the same gel and, respectively, lanes taken from different gels.