plx4032 was synthesized using the general procedures previously described.6 expression and purification of b - raf , structure determination , protein kinase activity measurements , and xenograft studies were carried out as previously described.6 clinical methods have also been recently described.5 melanoma patients were selected for study using previously described taqman methodology.8 semi - quantitative immunohistochemistry for perk and ki67 was performed on 5 m - thick formalin - fixed paraffin - embedded tumor biopsies following h&e staining to determine pathologic diagnosis and tissue morphology and integrity . the degree of phospho - erk staining in the nucleus and cytoplasm was interpreted semiquantitatively by assessing the intensity and extent of staining on the slides . for ki67 staining ,