FIG. 7.

(A) eIF2α-S51A can transform NIH 3T3 but not 3T3 L1 cell lines. Normal 3T3 L1 cells were transduced with retrovirus carrying the indicated genes. Stable colonies were selected following selection in G418. Morphology was assessed by phase-contrast microscopy. To measure transformation, two colonies per construct were selected and plated in 0.5% soft agar for 21 days. (B) Stable expression of eIF2α variants in 3T3 L1 cell lines was determined by Western blotting with a MAb directed against the FLAG tag. (C) NIH 3T3 cells were retrovirally transduced and assayed for transformation as described for panel A. (D) Stable expression of RasV12 or indicated eIF2α variants was confirmed by Western blotting with appropriate MAbs. Cloning efficiency for each cell line was determined following growth in soft agar for 21 days, by dividing the colony number by the total number of cells plated.