Fig. 5. Membrane targeting of WAVE2 is not sufficient for its regulation. (A) Confocal images of RAW/LR5 cells expressing either FLAG-tagged WAVE2 (left) or WAVE2CAAX (right) are shown with single XY, XZ and YZ cross-sections through different areas of the cell, as indicated by the cross-hair lines. Bar, 10 µm. (B) RAW/LR5 cells expressing either FLAG-tagged WAVE2 or WAVE2CAAX were fixed and stained for FLAG and F-actin and the total F-actin content was quantified as described in Materials and Methods and compared with nontransfected cells; n=3. (C) FLAG-tagged WAVE2CAAX-expressing RAW/LR5 cells were treated, or not, with CSF-1 for 5 minutes. The number of CSF-1-elicited protrusions was quantified as in Fig. 2D and expressed as a percentage of the CSF-1 stimulation observed in nontransfected cells on the same coverslip; n=3. (D) RAW/LR5 (white bars) and Cos-7 (gray bars) cells were either transfected with Myc-tagged Rac1Q61L (plain) or RacQ61L and FLAG-tagged WAVE2CAAX (striped) constructs. Coexpressing cells were identified by the Myc and FLAG staining, and the ability of cells to exhibit ruffles was quantified and expressed as a percentage of the total cells counted; n=3. (E) Mock- or WAVE2-shRNA-treated RAW/LR5 cells either transiently transfected with the indicated constructs, or not, were stimulated with CSF-1 and their ability to form F-actin-rich protrusions in response to CSF-1 was analyzed as in Fig. 2D and expressed as a percentage of the CSF-1 stimulation observed in nontransfected mock-shRNA-treated cells; n=3; ^*, P<0.05 compared with nontransfected mock-shRNA-treated cells (represented by the dotted line).