KSHV-infected cells express higher levels of B-Raf activity. (A) Target cells were cultured in low serum (1% FBS) containing medium for 12 hours and lysed with gold lysis buffer, and proteins were analyzed for B-Raf kinase activity by performing Raf kinase assay. Background radiation associated with nonspecific incorporation of [γ³²P]-ATP into MBP was determined with a control containing no B-Raf, MEK1, or MAPK/ERK2, and this value of 4662 cpm was deducted from the values obtained for all of the samples. To demonstrate the B-Raf–dependent activation of MBP via the MEK1, MAPK/ERK2 cascade, we performed a control in which no B-Raf protein or sample was added but MEK1 and MAPK/ERK2 were added. This control yielded an average of 2126 cpm. To demonstrate the MEK1-dependent MAPK activity, 1 ng of the recombinant B-Raf protein was added along with the MAPK/ERK2, as per the manufacturer's instruction, in the absence of MEK1. The MEK1-omitted sample yielded an average reading of 1966 cpm. Each reaction was done in triplicate, and each point represents the average ± SD of 3 experiments. (B) Western blot analysis of ERK1/2 expression in target cells. Cells were cultured in low serum containing medium for 12 hours, lysed as described above, and the proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The blots were probed for phospho ERK1/2 and total ERK1/2 by Western blotting. For all Western blots, β-actin levels demonstrated equal protein loading. (C) Western blot analysis after treating cells with inhibitor of the RAF/MEK/ERK signal transduction. Cells cultured in low serum containing medium were treated with different doses of PD98059 at 37°C. After 1 hour incubation, the cells were lysed as described above and probed for the expression of phospho ERK1/2 and total ERK1/2.