Fig. 6. Targeted depletion of P-Rex1 promotes spontaneous outgrowth of β-tubulin-rich projections (A) The location of the unique nucleotide sequence used to generate P-Rex1 RNAi clones is indicated in the P-Rex1 structure. (B) Cells were stably transfected with a plasmid encoding P-Rex1 RNAi sequence, or a scrambled RNAi control. Cell lysates (40 µg) were immunoblotted with P-Rex1 or actin antibodies. Relevant samples loaded on the same, representative immunoblot are shown. The relative P-Rex1 expression in P-Rex1 clones (1 and 5) was determined by densitometry of P-Rex1-immunoreactive polypeptides from five independent immunoblots, standardised to the actin loading control and expressed as a percentage of that detected in scrambled RNAi clones (3 and 4 combined). The bars indicate the mean ± s.e.m. of five independent experiments. (C) RT-PCR analysis of P-Rex1 RNAi clones. RT-PCR was performed on mRNA extracted from scrambled and P-Rex1 RNAi clones using Gapdh as a control. P-Rex1 expression was calculated and expressed relative to that from Scram(3), which was designated as one. Bars indicate the mean ± s.e.m. of three independent experiments. (D) Cells stably transfected with P-Rex1 RNAi or scrambled RNAi control were left unstimulated (a,b,c) or NGF-stimulated for 10 minutes (d). Cells were stained with Texas Red-conjugated phalloidin (red or grey) or β-tubulin antibody (green) to image cell morphology. Representative images of unstimulated P-Rex1 RNAi clones (1 and 5) and scrambled RNAi control (4) are shown (a,b) with merged magnified images of boxed regions (c). β-tubulin-containing projections are indicated (b, arrows). Merged images of cells treated with NGF for 10 minutes are shown (d) with areas of extensive peripheral F-actin indicated by arrowheads. Scale bars: 5 µm. (E) RNAi-mediated depletion of P-Rex1 in PC12 cells reduces Rac3 activation in response to NGF stimulation. Scrambled RNAi or P-Rex1 PC12 RNAi clones were transiently transfected with myc-Rac1 or HA-Rac3, serum starved and NGF-stimulated (3 minutes). Duplicate samples of cell lysates were subjected to colorimetric Rac1,2,3 G-LISA Activation Assay and the average Rac activation was calculated relative to serum-starved scrambled control cells. Equivalent expression of Rac constructs was confirmed by immunoblot analysis using a pan-Rac antibody (not shown). Bars indicate mean ± s.e.m. of relative Rac activation for four independent experiments. ^**P<0.005; ^***P<0.001.