Nilotinib leads to BRAF-CRAF heterodimerization and subsequent MEK-dependent ERK activation in head and neck squamous cell carcinoma (HNSCC) cells. (A) HNSCC cells were treated with 1 μmol/L nilotinib for the indicated times and then lysed. RAF dimerization was measured using immunoprecipitation (IP) with an anti-CRAF antibody followed by immunoblotting for total BRAF and CRAF (upper panels). To check MEK and ERK activation, we subjected lysates to immunoblotting with the indicated antibodies (lower panels). (B) Cells were treated with 1 μmol/L nilotinib, 150 nmol/L MEK162, or both for 3 hours and lysed. Cell lysates were resolved by SDS PAGE and Western blot analysis was performed to measure levels of pMEK, pERK, total MEK, ERK, and μ-actin.