Figure 4
A) Up-regulation of Zizimin2 in response to stimulations. 107cells/ml of BMDC without or with stimulation by 1 μg/ml anti-IgG Fab'2 for 2 min (left panel, anti-FcγRII/III), 1 μg/ml LPS for 5 min (middle panel), or 1 μg/ml anti-CD40 for 3 min (right panel) were lysed and subjected to western blotting. α-Tubulin was used as a loading control. B) Cdc42 activation in response to stimulations. 107cells/ml of BMDC were stimulated with 1 μg/ml anti-IgG Fab'2 for 0 to 10 min (left panel, anti-FcγRII/III) or 1 μg/ml LPS for 0 to 30 min (right panel) and subjected to Cdc42 activation assay. Activated Cdc42 was assayed by precipitating GTP-loaded Cdc42 with bead-bound GST-PAK1-PBD, and detection of Cdc42 in pull downs (upper panel) or total cell lysates (lower panel) by western blotting with anti-Cdc42. C) Filopodial formation in BMDC. BMDC without or with stimulation by 1 μg/ml anti-IgG Fab'2 for 5 min (anti-FcγR) or 1 μg/ml LPS for 5 min were stained with phalloidin, which was bound with F-actin (left panel, green), anti-mouse-Zizimin2 antibody, and anti-rat-IgG-Cy3-conjugated antibody (middle panel, magenta) followed by immunofluorescence microscopy. Right panels are merged images of detected phalloidin (Alexa488) and detected Zizimin2 (Cy3). White bars are 100 μm.