|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 4. Migration-induced activation of Rac1 is reduced in RhoG RNAi HeLa cells. (A) Phase contrast images of wound-healing assays of parental, control RNAi and RhoG RNAi HeLa cells at 0 and 9 hours after wounding. Cells were plated in complete medium at a confluent density and scratched with a micropipette tip. Average rates of wound closure were calculated from three independent experiments. Bar, 300 µm. (B) Cells were fixed 2 hours after wounding and stained with Alexa Fluor 594-conjugated phalloidin to examine cell morphology at the wound edge. Bar, 20 µm. Cells extending lamellipodia into the open wound area were scored as a percentage of the total number of cells at the wound edge, and data are the means ± s.e.m. of three independent experiments. (C) RhoG RNAi HeLa cells transiently transfected with GFP or GFP-tagged mouse RhoG (GFP-mRhoG) were fixed 2 hours after wounding and stained with Alexa Fluor 594-conjugated phalloidin to examine cell morphology at the wound edge. Bar, 20 µm. (D) Multiple scratch wounds were made in confluent cells, and GTP-bound Rac1 in the cells at indicated times after wounding were precipitated with the GST-fused CRIB domain of Pak. The amounts of GTP-bound Rac1 and total Rac1 were detected by immunoblotting with anti-Rac1 antibody. Relative Rac1 activity was determined by the amount of GTP-bound Rac1 bound to GST-CRIB normalized to the amount of Rac1 in cell lysates analyzed by NIH Image software, and the value from control RNAi HeLa cells at 0 minute after wounding was arbitrarily set at 1. Data are the means ± s.e.m. of three independent experiments.
|
