Fig. 4. IRSp53 exists in a complex with WAVE2 and Abi1 in a Rac1-dependent manner. (A) Lysates from nontransfected or GFP-tagged IRSp53 and Myc-tagged Rac1- or Rac1N17- or Rac1Q61L- or Cdc42V12-coexpressing RAW/LR5 cells and Cos7 cells were immunoprecipitated with antibodies against either GFP or Myc (IP) and subjected to western blotting using the indicated antibodies. A representative example of three independent experiments is shown. (B) Lysates from mock- or IRSp53-shRNA-treated RAW/LR5 cells expressing Myc-tagged Rac1Q61L were immunoprecipitated with antibodies against Myc (IP) and were then subjected to western blotting using the indicated antibodies. (C) Quantification of WAVE2 or Abi1 co-immunoprecipitated by Myc (from B) is shown; n=3; ^*, P<0.05 compared with mock-shRNA-treated cells expressing Rac1Q61L. (D) Lysates from nontransfected or Myc-tagged Rac1Q61L- and HA-tagged IRSp53SH3-coexpressing RAW/LR5 cells were incubated with antibodies against HA for immunoprecipitation (IP-HA), followed by sequential immunoprecipitation of Rac1Q61L using antibodies against Myc (IP-Myc). Immunoprecipitates were then subjected to western blotting using the indicated antibodies. Mock- or IRSp53-shRNA-treated (E) or WAVE2-shRNA-treated (F) 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).