Fig. 7. The effect of overexpressing wild-type or S36A mutant p66Shc on focus formation and enhanced growth of HDFs. (A) HDFs stably co-expressing wild-type (WT) p66Shc, S36A or the corresponding empty retroviral expression vector (LXSN) with E5, v-Sis or no viral oncogene (Puro) were seeded at an equal density in a 24-well dish. At various times thereafter, cells were trypsinized and counted in triplicate. The mean cell number with standard error is plotted against the time after plating. (B) Whole-cell extracts of cells co-expressing the indicated transgenes were subjected to immunoblot analysis for Ser36 phosphorylated p66Shc (PS³⁶-p66Shc), or phosphorylated AKT or JNK (pAKT or pJNK, respectively). Blots were reprobed for total p66Shc, AKT or JNK. JNK isoforms 1 and 2 are indicated. (C) A focus-forming assay was performed by infecting HDFs expressing wild-type p66Shc, S36A or no transgene (LXSN) with E5- or v-Sis-containing retrovirus. Foci were counted and averaged from several experiments. Focus formation is expressed as the mean percent (with standard error) relative to the number of foci formed by the LXSN-HDFs.