FIG. 4.

MAPK is constitutively phosphorylated and activated in SFFV-infected cells. (A) Uninfected and SFFV-infected HCD-57 cells starved overnight in 1.5% FCS and either left stimulated (−) or stimulated with Epo for 15 min (+) were resuspended in SDS lysis buffer and analyzed by Western blot analysis using a phosphospecific anti-MAPK (p42/44) antibody to detect tyrosine-phosphorylated MAPK (MAPK-PTYR). The blot was then stripped and reprobed with a p42/44-specific anti-MAPK antibody that detects both the phosphorylated and unphosphorylated forms of MAPK. (B) Immunoprecipitated MAPK was assayed in immune complex kinase assays measuring incorporation of ³²P into MBP. Phosphorylation of MBP was detected by autoradiography, and the filter was then probed with anti-MAPK antibody to visualize the amount of MAPK present in the kinase reaction. IgG, immunoglobulin G.