Figure 7. KAI1/CD82 inhibits ROCK activity and the enrichment of RhoA at the cell periphery.

(A). Du145-Mock and -KAI1/CD82 transfectant cells were cultured in either DMEM containing 0.5% FBS for 40 h or DMEM containing 10% FBS and HGF (100 ng/ml) for overnight. The cells were then lysed with 1% Triton X-100 lysis buffer. Equal amounts of cell lysate were assayed for ROCK activity with MBL Rho Kinase Assay kit. In each experiment, ROCK activities were measured in duplicate for each transfectant. The results shown in histogram are the average values of three individual experiments±SD. *: P<0.05, **: P<0.01. (B). The cells were pretreated with HGF (100 ng/ml) overnight and then assayed for RhoA activity as aforementioned. Histogram represents the relative density of the RhoA-GTP bands (mean±SD, n = 3). *: P<0.05. (C). The cells spread on LN1 (20 µg/ml)-coated plates were treated with or without HGF (100 ng/ml) overnight and then fixed, permeabilized, and incubated sequentially with RhoA mAb and Alexa594-conjugated second Ab. Images were acquired as described above under a fluorescent microscope. Arrows indicate the enrichment of RhoA at the cell periphery and in the retraction tail. The bottom panel shows the fluorescent profiles of line scan from Mock and KAI1/CD82 cells.