Figure 1.
PIAS3 is a new Rac1-binding protein required for optimal Rac activation and cell migration in response to HGF. (a) GST protein alone or GST-Rac1 were incubated with cell extracts containing FLAG-PIAS3 and protein pulldowns (PD) were analyzed by Western blot (WB). (b) Epitope-tagged proteins were expressed in HEK293T cells and GFP immunoprecipitation (IP) performed. Purified proteins were detected by WB. IN, input. (c) GST-Rac1 beads divided in two samples were incubated with GDP or γGTP for 1h at 30°C and then with equal amounts of cell lysate containing FLAG-PIAS3 for 1 hour at 4°C. PIAS3 interaction was detected by WB. (d) HEK293T cells were transfected with the indicated plasmids and subjected to GFP immunoprecipitation. Samples were analyzed by WB. (e) Individual cells were tracked for at least 24 hours in the presence of HGF. Data shown is average speed of migration of cells from at least three independent experiments (at least 30 cells per experiment). *p<0.001, ** p<0.05, ns indicates no significant difference, two-tailed Student’s t-test. Values are means ± standard deviation. (f) MDCKII cells inducibly expressing pias3 or scr shRNAs were treated with doxycycline (dox), serum was then removed for 18 hours before cells were treated with HGF for 30 minutes and Rac activity measured. Bands were quantified and normalized intensities were calculated relative to control. Values are means ± standard deviation (n=3). (g) MDCKII cells inducibly expressing pias3 or scr shRNAs were treated as above but Rac activity was measured at later time points. (h) COS7 cells transfected with MYC-PIAS3 or empty vector were serum starved for 18 hours before 10 ng/ml of HGF was added for 30 minutes and Rac activity measured. (i) Bar chart shows quantification of the normalized relative amounts of Rac-GTP of (h) determined by scanning densitometry. Error bars indicate standard deviation for 3 independent experiments. ***p<0.001, two-tailed Student’s t-test. Uncropped images of blots are shown in Supplementary Information, Fig. S6.