P n wasps484 485

T-47D cells were grown on coverslips. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton for 5 min. Blocking was performed with PBS containing 3% bovine serum albumin for 30 min at room temperature. Cells were incubated with antibodies against p-paxillin^Y118; vimentin (E-5) (Santa Cruz Biotechnology); E-cadherin (24E10) (Cell Signaling Technology); p-N-WASP^S484/485 (Chemicon International); and p-Arp2^T237 (Biorbyt) overnight at 4 °C, followed by incubation with DyLight⁴⁸⁸/ DyLight⁵⁹⁴ and/or fluorescein-conjugated secondary antibody (FITC 1:150; Vector Laboratories, Burlingame, CA). Cells were then incubated with Texas Red-phalloidin (Sigma-Aldrich, Saint-Louis, MO) for 30 min. After washing, the nuclei were counterstained with or 4'-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Saint-Louis, MO) and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence was visualized using a Nikon Eclipse E200 microscope and recorded with a high-resolution DP70 Olympus digital camera.

T-47D cells were grown on coverslips and exposed to different treatments. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton for 5 min. Blocking was performed with phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) for 30 min at room temperature. Cells were incubated with antibodies against p-N-WASP^S484/485 (Chemicon International) and p-paxillin^Y118 and p-cortactin^Y466 (Santa Cruz Biotechnology) overnight at 4°C, followed by incubation with DyLight 594 and/or fluorescein-conjugated secondary antibody (FITC 1:150; Vector Laboratories). Cells were then incubated with Texas Red–phalloidin (Sigma-Aldrich) for 30 min. After washing, the nuclei were counterstained with 4’-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) and mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence was visualized using a Nikon Eclipse E200 microscope and recorded with a high-resolution DP70 Olympus digital camera.

Cell lysates were separated by SDS-PAGE in 8–10% gels and transferred into polyvinylidene difluoride (PVDF) membranes. Antibodies used were p-FAK^Y397 (611807), FAK (610088), and Arp3 (612135) (BD Transduction Laboratories); actin (sc-1615), LHR (sc-25828), c-Src (sc-5266), paxillin (sc-31010), p-paxillin^Y118 (sc-365020), cortactin (sc-11408), p-cortactin^Y466 (sc-101611), N-WASP (sc-13139), Arp2 (sc-15389), and p-Tyr (sc-7020) (Santa Cruz Biotechnology); p-N-WASP^S484/485 (ab1964) (Chemicon International); p-Src^Y418 (ab4816) (Abcam); and p-Arp2^T237 (orb155730) (Biorbyt). Primary and secondary antibodies were incubated with the membranes using standard techniques. Immunodetection was accomplished using enhanced chemiluminescence and recorded with a quantitative digital imaging system (ChemiDoc XRS with Image Lab, Bio-Rad).