Figure 4.

RhoA activation depends on PI3Kγ and Cdc42. (A) fMLP-induced membrane association of RhoA. Cells received no pretreatment or were exposed for 40 min to 0.5 or 1 μM of PIK-90 as indicated and were stimulated with or without 100 nM fMLP for 1 min as indicated. RhoA that associated with the particulate fraction of cell extracts was then assessed as described in Materials and methods. The top and bottom panels show a representative immunoblot and quantification of immunoblotted bands measured with the Image program (Scion), respectively. For each immunoblot, the background signal was subtracted, and the level of RhoA was normalized to that of transferrin in the particulate fraction; all values were further normalized to the signal detected in the absence of inhibitor, which was set at 1.0. Each graph represents the mean of three independent experiments, with different symbols representing the three actual values. (B–G) Quantification of mean FRET/CFP ratios in cells treated under different conditions. Data were obtained from n ≥ 25, 14, 18, 12, 13, or 13 cells in B–G, respectively, and were normalized to the FRET/CFP signal measured in unstimulated control cells. Similar results were observed in three independent experiments. Error bars indicate one SEM. t tests were performed to compare data of unstimulated control versus other experimental groups and between stimulated control and other conditions. Pairs of treatments that showed a statistically significant difference are marked with asterisks (*, P ≤ 0.05; **, P ≤ 0.001 by t test). (B) Lentivirus-transformed cells expressing the RhoA biosensor were treated with or without 1 μM PIK-90 for 40 min, stimulated with 100 nM fMLP for 3 min as indicated, fixed, and subjected to FRET imaging. (C–G). dHL60 cells transiently transfected with the RhoA biosensor and Cdc42-V12, ΔC-WASp, Rac-V12, Rac-N17, or G12/G12-DN, as indicated, were stimulated with 100 nM fMLP for 3 min as indicated.