Dominant negative RhoA inhibits membrane ruffling and lamellae formation in clone A cells in response to laminin-1. Clone A cells were cotransfected with a GFP construct and either a control vector or a vector encoding N19RhoA or N17Rac as described in Materials and Methods. Cells were plated onto laminin-coated coverslips for 40 min, fixed, and assessed by phase-contrast microscopy. A–C, Phase-contrast microscopy of vector control (A), N19RhoA (B, two panels), or N17Rac (C) transfected cells. Note large lamellae and membrane ruffles in control and N17Rac transfected cells (open arrow in A and C), but not in cells that express N19RhoA (B). Representative GFP-positive cells are shown. D, Quantitative analysis of the lamellar area of transfected, GFP-positive cells was obtained by digital imaging. Lamellae are defined as broad, flat cellular protrusions rich in F-actin and devoid of membrane-bound vesicles. E, Quantitative analysis of total area covered by cells transfected with either vector control or N19RhoA when plated on laminin-1 (dark bars) or collagen I (light bars). Bars represent mean area ± SEM in which n > 20 (D, E). F and G, Transfected cells were extracted with RIPA buffer and either immunoprecipitated with HA-specific mAb and immunoblotted for RhoA (F), or concentrated using glutathione-Sepharose and immunoblotted for Rac1 (G). Representative blots are shown.