Figure 4.

PIP5K-γ−/− BMM have abnormal RhoA and Rac1 activation. (A) GTP-Rac1 or -RhoA pull down by GST-PBD or -RBD, respectively. Samples were blotted with anti-Rac1 or -RhoA. (left) Western blot. Three times more WT BMM lysate and GST-RBD pull-down sample were loaded than PIP5K-γ−/− BMM. (right) Ratios of GTP-bound to total GTPase, expressed relative to the levels of WT BMM (n = 4 for Rac1 and 2 for RhoA). (B) Rescue of particle binding defect by C3T. BMM were incubated with C3T at 37°C for 4 h before the exposure of IgG-opsonized particles at 4°C. (left) Fluorescence staining. Green, external beads; red, phalloidin. (right) Binding indices (n > 70). (C) Effects of manipulating Rac activation. BMM were transduced with 600 nM Tat-Rac1L61 or N17 at 37°C for 30 min. (left) Fluorescence images. Green, external beads; red, phalloidin. Arrowheads indicate dorsal ruffles. (top right) Particle binding indices (n > 50). (bottom right) Fluorometric phalloidin quantitation (n = 3). Data are expressed as the percentage of WT BMM without transduced Rac1. Error bars indicate SEM. Bars, 10 µm.