Fig. 3

MIIP reduces formation of lamellipodia, affects Rac1 location, and inhibits Rac1 activity. a HEC1A cells were transfected with si-control or si-MIIP(#1and #2) for 72 h and then stained with phalloidin. Arrow: lamellipodia. b HEC1B cells were infected with Ad-MIIP or control adenoviral vector (Ad-Ev) at 10 multiplicity of infection (MOI) for 48 h and then stained with phalloidin (red). Arrow: lamellipodia. c HEC1A cells were transfected with si-control or si-MIIP (#1and #2) for 72 h and then stained with Rac1. Arrow in the upper panel: Rac1 location. Arrow in the middle and lower panels: Lamellipodia and Rac1 location. d HEC1B cells were infected with Ad-MIIP or Ad-Ev at 10 MOI for 48 h and then stained for Rac1(red). Arrow in the upper panel: Lamellipodia and Rac1 location. Arrow in the lower panels: Rac1 location. All images were taken by ZEISS HEO 100 microscope at a magnification of ×630. DNA was stained by DAPI (blue). Scale bar 10 μm. e Rac1 activity assay was based on the level of Rac1-GTP pulled down by the PAK1 PBD. HEC1A cells were transfected with MIIP siRNA or scramble control siRNA. GTP-Rac1 immunoprecipitated by GST-PAK1 PBD was detected by western blotting with an anti-Rac1-specific antibody. The left bar chart shows relative levels of MIIP, and the right bar chart shows relative levels of Rac1-GTP. Rac1 activity is presented as percentage of GTP-Rac1 compared to total Rac1 (as 100 %). Data are shown as means ± SD from three independent experiments; *P < 0.01 (t test). f Rac1 activity assay was performed on HEC1B cells overexpressing MIIP. HEC1B cells were infected with Ad-MIIP or control adenoviral vector (Ad-Ev). GTP-Rac1 immunoprecipitated by GST PAK1 PBD domain was detected by western blotting with Rac1 antibody. MIIP was also pulled down by PAK1. The bar chart shows relative Rac1 activity. Data are shown as means ± SD from three independent experiments; *P < 0.01 (t test)