FIG. 8.

Mutation of Ser64 and/or Thr189 impairs the ability of C/EBPβ to cooperate with Ras^V12 in NIH 3T3 focus assays. (A) NIH 3T3 cells were transfected with 10 ng each of the C/EBPβ and Ras^V12 expression vectors and scored 2 weeks later for the presence of transformed foci, as described previously (7). Transfections were performed in triplicate, and the data were averaged (± standard deviations); results from four independent experiments are shown. Transfection of C/EBPβ alone produced no transformed foci. *, P < 0.01 relative to Ha-Ras^V12 (by Student's t test). (B) NIH 3T3 cells were transfected with expression vectors for Ras^V12 and the indicated C/EBPβ protein. The cells were serum starved for 16 h, RIPA buffer lysates were prepared, and 25 μg of each extract was analyzed for C/EBPβ expression by Western blotting. (C) Extracts from panel B were analyzed by an electrophoretic mobility shift assay using equivalent amounts of total protein in binding reactions containing a radiolabeled C/EBP binding site probe. Extracts from mock-transfected cells (lane 2) and Ras^V12-transfected cells (lane 8) were included as controls. A C/EBPβ antibody supershift (lane 7) shows that all of the observed DNA-binding species contain C/EBPβ.