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Fig. 3. Ectopic expression of P-Rex1 inhibits NGF-mediated neurite differentiation and is dependent on both the Rac-GEF activity and the 4-phosphatase domain. (A) Schematic of P-Rex1 deletion mutant constructs. PC12 cells were transiently transfected with P-Rex1 mutant constructs and cell lysates were immunoblotted with HA or myc antibodies, as shown beneath. 1, HA-vector; 2, HA-P-Rex1; 3, HA-P-Rex1N; 4, HA-P-Rex14P; 5, myc-P-Rex1; 6, myc-P-Rex1GEFdead. To the right, neurite outgrowth, neurite F-actin and initiation are summarised for each P-Rex1 mutant protein. For neurite outgrowth: +++, as vector control; +, reduced neurite outgrowth; –, no outgrowth. For F-actin: +, as vector control; +++, increased. For initiation: +, initiation as vector control. (B,C) PC12 cells were transiently transfected with wild-type or mutant P-Rex1 (5 µg, unless otherwise indicated) and NGF-stimulated for 3 days in the presence or absence of 1 µM cytochalasin D. (B) Cells were co-stained with HA or myc antibodies (green) and Texas Red-conjugated phalloidin (red or grey). Merged images are shown (lower row) with neurites or actin-rich projections indicated by arrows. Scale bar: 10 µm. (C) Cells containing actin-rich projections were imaged and the length of the neurite/projection and the cell body diameter determined. Bars indicate the mean ± s.e.m. of cells bearing neurites longer than one cell body diameter. 100 cells were scored for each construct for three independent transfections. ^*P<0.05; ^**P<0.01; ^***P<0.001.

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