Fig. 3

The GAS7b is localized in cell periphery and interacts with CYFIP1 to suppress the binding of Rac1 active form. a MDA-MB-231 cells were transfected with HA-tagged-GAS7b expressing plasmid. The cells were plated on fibronectin coated coverslips for 90 min, and confocal microscopy images of Paxillin (green) for FA, HA (purple) for GAS7b, and DAPI (blue) for nuclear staining were taken. b The MDA-MB-231 cells expressing HA-tagged-GAS7b were stained with anti-HA (green) antibody, Phalloidin (red), and DAPI (blue), and images from confocal microscopy were taken. The Phalloidin staining for F-actin that zoom in 4× are also shown. c Co-immunoprecipitation (co-IP) assay with MCF-7 cells was carried out. Anti-GAS7 and anti-CYFIP1 antibodies were used for IP and western blotting. Normal goat IgG was used as the negative control for IP experiments. d MCF-7 cells were transfected with control or GAS7 siRNA, and subsequently super-transfected with the constitutively active Rac1 (ca-Rac1) plasmid. Anti-CYFIP1 was used in the IP assay to pulldown associated proteins, anti-CYFIP1 and anti-Rac1 antibodies were used for western blotting. Normal rabbit IgG was used as the negative control