Fig. 4. P-Rex1 co-localises with, and promotes the activation of, HA-Rac3 in NGF-stimulated PC12 cells. (A) PC12 cells were co-transfected with myc-Rac1 or HA-Rac3 and vector control, P-Rex1 or P-Rex1GEFdead, and left serum starved or stimulated with NGF for 3 minutes. Duplicate samples of cell lysates were subjected to colorimetric Rac1,2,3 G-LISA Activation Assays and the average Rac activation was calculated relative to serum-starved HA-vector-only controls. 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<0.05). (B) PC12 cells were transfected with HA-P-Rex1 (upper row) or co-transfected with HA-P-Rex1 and myc-Rac1 (middle row) or myc-P-Rex1 and HA-Rac3 (bottom row). Cells were stimulated with NGF for 3 minutes. P-Rex1 (green) and Myc-Rac1 or HA-Rac3 (red) were detected using antibodies to each tag. Co-localisation is indicated by yellow staining in the merged images (right-hand column). Arrowheads indicate P-Rex1/Rac1 membrane co-localisation, with P-Rex1/Rac3 perinuclear co-staining indicated by the arrow. Scale bars: 10 µm.