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Fig. 7. P-Rex1 regulates NGF-stimulated neurite elongation. Cells stably transfected with P-Rex1 RNAi, or a scrambled control RNAi, were NGF-stimulated for 3 days and stained with Texas Red-conjugated phalloidin and β-tubulin antibodies to image neurite morphology. (A) Representative images of NGF-differentiated P-Rex1-depleted or control PC12 cells. Scale bars: 10 µm. (B) The numbers of differentiated PC12 cells with short neurites (length less than two cell body diameters) and long neurites (length greater than three cell body diameters) were expressed as a percentage of all differentiated neurites. Bars indicate mean ± s.e.m. of at least 100 cells scored for each of three independent differentiation experiments. (C) Neurite length was expressed as the fold increase (mean ± s.e.m.) of the length of the longest neurite for P-Rex1 clones 1 and 5 relative to that of the scrambled control (4). At least 60 neurites were scored for each of three independent differentiation experiments. (D) P-Rex1 RNAi clone (5) was transiently transfected with HA empty vector or with plasmids containing HA-P-Rex1 (1, 2 or 5 µg of DNA), myc-P-Rex1GEFdead (5 µg), HA-P-Rex1N (5 µg) or HA-P-Rex14P (5 µg) and NGF-differentiated for 3 days. Cells were stained with Texas Red-conjugated phalloidin and HA or myc antibodies. The number of cells bearing neurites longer than two cell body diameters was calculated and standardised relative to that of the P-Rex1 RNAi clone (5). Knockdown cells transfected with HA empty vector were scored as 100%. Bars indicate the mean ± s.e.m. for at least 50 cells scored per indicated construct for each of three independent experiments. ^*P<0.05; ^**P<0.01; ^***P<0.001.

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