FIG. 5.

PI(4,5)P₂ production in PC12 cells expressing v-Crk and v-Crk mutants. Native PC12 cells, v-Crk cells (clones V15F and V1F), R273N–v-Crk cells, or D386DRHAD–v-Crk cells were maintained in 15% serum, after which they were briefly starved and incubated overnight with 20 μCi of [³²P]orthophosphate. Extracts were prepared from adherent cells and normalized for cellular protein, and the resulting radiolabeled lipids were subjected to TLC. ³²P-labeled PIP₂ was deacylated and quantified with a PhosphorImager. Values were normalized to the value in native PC12 cells (designated 100%) and are expressed as the mean and standard error (P < 0.05 between V15F and V1F and native cells, indicated by asterisk) of four independent experiments. HPLC analysis on extracted lipids demonstrated that only PI(4,5)P₂, and not PI(3,4)P₂, was produced in these cells.