Supplemental Material

This article contains the following supporting material:

• Figure 1 - The extension of neurites that is induced by activated H-Ras requires ERK MAP kinase activation but not the PI3K/Akt pathway. Expression of H-Ras.V12 produces prominent neurite extensions in approximately 50% of the transfected PC12 cells within 48 hours (upper left: arrows indicate cells with neurites; arrowheads indicate cells without obvious morphological differentiation). Representative panels show that neurite extension is reduced by treatment with U0126 (upper middle) or co-expression of dominant-negative MEK1 (lower middle), but is unaffected by treatment with wortmannin (upper right) or SP600125 (lower left) or coexpression of dominant-negative Akt (lower right). Scale bar = 100 μm.

• Figure 2 - Activated Rac1 induces membrane ruffling through a pathway that requires PI3K and Akt activity. (A) Representative results show that expression of constitutively activated Rac1.V12 induces prominent membrane ruffles (upper left) that are inhibited by either treatment with wortmannin or co-expression of dominant-negative Akt (upper middle panels). The Rac1.V12 phenotype is maintained in the presence of H-Ras.V12 (upper right) or dominant-negative MEK1 (lower left) or treatment with SP600125 (middle bottom), which also does not prevent the morphological differentiation induced by Ras-GRF1 plus H-Ras (lower right). (B) Images of a representative, triply co-transfected PC12 cell that shows that the Rac1.V12 phenotype is also maintained with co-expression of GRFΔN plus H-Ras. Scale bar = 50 μm.