|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 9. Aberrant localization of PI3K in sodC– cells. (A) The membrane localization domain of PI3K1 (N-PI3K1) fused with GFP was expressed in wild-type and sodC– cells. Aggregation-competent polarized wild-type cells clearly demonstrated localized PI3K membrane translocation at the leading edge, whereas sodC– cells showed no PI3K polarization around the membrane. By contrast, GFP-PTEN localization was indistinguishable between wild-type and sodC– cells. (B) 0.01% Triton X100 fraction showed that more N-PI3K1-GFP proteins were aberrantly enriched in the membrane fraction of sodC– cells than of wild-type cells. (C) Cells expressing PI3K1-LD-GFP proteins were pulsed with 50 nM cAMP for 4 hours, and stimulated with 10 µM cAMP. Membrane translocation of PI3K1-LD protein was recorded at 10-second intervals. Clear membrane localization of PI3K1-LD was observed in wild-type cells, but no such changes were seen in sodC– cells. (D) Cells were pulsed for 4 hours, fixed and stained with TRITC-phalloidin, as described in the Materials and Methods. Two representative images are shown for each wild-type and sodC– cell. Wild-type cells displayed more polarized cell bodies with a leading edge enriched with F-Actin. By contrast, sodC– cells were much more round than the wild type and showed numerous filopodia-like structures instead of a dominant pseudopodium. (E) Pulsed cells were stimulated with 10 µM cAMP as indicated, and lysed with a F-Actin buffer containing 0.2 % of Triton X-100 and TRITC-phalloidin, and the F-Actin levels were measured as described in the Materials and Methods. sodC– cells displayed a higher basal level of F-Actin compared with wild type, but no wild-type-like response was observed after cAMP stimulation.
|
