Figure 5

Knockdown of GRASP inhibits HGF induced migration and Rac activation in MDCK cells. A) GRASP levels in MDCK cells were reduced by transfection of siRNA and cell migration measured using the Oris migration chamber. B) Cell migration was quantitated by measuring the percent of the starting open area covered. Data shown are the mean ± standard error of at least 15 separate samples. C) GRASP knockdown and control cells were allowed to migrate in a transwell chamber toward 10 ng/ml HGF. Data shown are the mean ± standard error of at least 15 separate experiments. Migration of the knockdown and control cells in both B. and C was compared using a standard t-test. ** p < 0.01. D) MDCK cells were transfected with control or GRASP-targeting siRNA as described in Experimental Procedures. After 48 hours mRNA was isolated and RT-PCR of the canine GRASP or GAPDH performed. E) MDCK cells were transfected with siRNAs and 24 hours later were split onto duplicate plates. The next day the cells were incubated in the presence or absence of HGF for 5 hours and active Rac isolated by binding to GST-PBD. Rac levels in saved aliquots of the starting lysates and the isolated Rac-GTP were detected by Western blotting. F) Rac activation levels in 4 independent knockdown experiments were normalized to the level of active Rac in the control 0 ng/ml HGF sample. Rac activation in the HGF stimulated control and GRASP knockdown samples were compared using a paired t Test. Data shown are mean ± standard error. * = p < 0.05. G) MDCK cells were transfected with control or GRASP siRNAs. After 48 hours the cells were treated with 20 ng/ml HGF for the indicated times, harvested and blotted for phosphorylated cMet and total cMet.