The cellular localization of the IRAS protein was identified according to previously described procedures [27] . Cells on glass cover slips were rinsed with PBS, fixed with 3% paraformaldehyde for 30 minutes, and permeabilized with 0.2% [v/v] Triton X-100/PBS. Permeabilized cells were incubated with IRAS mAbs and TRITC-conjugated affinipure goat antimouse by PCR amplification of the human IRAS cDNA (AF082516) using the following oligomers: sense, 5 -CGGGATCCA-TGGAGCGGGAAGCCGAGCCG-3 , and antisense, 5 -CGGAATTCATAGAAGTGAAAATGCAAGAAGTG-3 . The full-length human IRAS was obtained by PCR amplification of the entire coding region, and the resulting 4512-bp PCR product was ligated into the pEGPC1 and PCMV-myc vectors in a similar fashion using the following oligomers, respectively: sense, 5 -CGCGAATTCTATGGCGACCGC-GCGCACCTTCG-3 , and antisense, 5 -CGGGATCCTAGC-CGGTGAGCTCGACAGGC-3 , sense, 5 -CGCGAATTC-GGATGGCGACCGCGCGCACCTTC-3 , and antisense, 5 -CCGCTCGAGCTAGCCGGTGAGCTCGACAGGC-3 . All plasmid sequences were confirmed by sequencing analysis.