FIG. 2.

Infection with SFFV leads to constitutive activation of Raf-1 in HCD-57 cells. Raf-1 immunoprecipitates from uninfected and SFFV-infected HCD-57 cells that had been starved overnight in 1.5% FCS and either left unstimulated (−) or stimulated with Epo for 15 min (+) were assayed for Raf-1 kinase activity. (A) Immune complex kinase assay measuring incorporation of ³²P into GST-MEK⁻. Phosphorylation of MEK⁻ was detected by autoradiography. The filter was then probed with an anti-Raf-1 antibody to visualize the total amount of Raf-1 present in the kinase reaction. (B) Coupled-kinase assay measuring Raf-1 activation of MAPK, using exogenous GST-MEK and GST-MAPK as substrates. Activation of GST-MAPK by Raf-1 was assayed by phosphorylation of MBP. Specific incorporation of ³²P into MBP was measured by scintillation counting. Results are reported after substraction of values obtained from control reactions in which either the MEK substrate or Raf-1 was not included in the reaction mixture. The amount of Raf-1 in the assay was visualized by Western blot analysis of the immunoprecipitated protein with anti-Raf-1 antibody.