Journal: Cell Death & Disease
Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion
Figure Lengend Snippet: PAK1 can associate with RUFY3 in vitro and in vivo. ( a ) PAK1 phosphorylates RUFY3 in vitro . Glutathione S -transferase (GST) and GST-RUFY3 were used as PAK1 substrates in PAK1 kinase assay. ( b ) A specific interaction between RUFY3 and PAK1 was demonstrated by in vitro GST assay. Asterisks indicate GST, GST-RUFY3 and GST-PAK1 bands, and ponceau staining indicates the loading amounts. (Left panel) In vitro -translated His-PAK1 binds to purified GST-RUFY3. (Right panel) In-vitro -translated His-RUFY3 specifically interacts with GST-PAK1. ( c ) GFP-RUFY3 was co-precipitated with myc-PAK1. The cell lysate from HEK293 cells transfected with myc-PAK1 and GFP-RUFY3 were incubated with anti-GFP antibody. The immunoprecipitates were analyzed by western blot with anti-myc and anti-GFP antibodies. ( d ) Co-IP assays to identify endogenous PAK1 interacting with endogenous RUFY3 in COS-7 cells (left panel) and BGC-823 cells (right panel). In vivo anti-PAK1 antibody or anti-RUFY3 antibody immunoprecipitates endogenous RUFY3 or PAK1. Immunoblots were carried out as indicated. ( e ) Colocalization of PAK1 with GFP-RUFY3 at the cell periphery is shown by confocal microscopy. SGC-7901 cells were transiently transfected with GFP vector or GFP-RUFY3. (Left panel) Colocalization of PAK1 (red) with GFP-RUFY3 is shown by yellow fluorescence. Scale bars, 10 μ m. (Right panel) Histogram showed the relative percentage of colocalization cells expressing GFP-RUFY3 with PAK1 at the cell periphery. The data show mean±S.E.M. (** P
Article Snippet: Immunofluorescence, time-lapse image acquisition and confocal microscopy analysis SGC7901 cells transfected with GFP vector or GFP-RUFY3 grown on glass coverslips were fixed in methanol at room temperature for 15 min, and then blocked with normal goat serum for 1 h. The cells were incubated with rhodamine-conjugated phalloidin (Sigma) to detect F-actin for 1 h at room temperature; rabbit anti-integrin α 3β 1 (1 : 50; Bioss Inc.), mouse anti-integrin α 3β 1 (1 : 100; Abcam); rabbit anti-vinculin (1 : 100; Santa Cruz, Santa Cruz, CA, USA), rabbit anti-myosinIIb, integrin β 5 and Flag-tagged (1 : 100; Shanghai, Kangcheng, Shanghai, China), PAK1 (1 : 50; Cell Signaling) antibody were used overnight at 4 °C and rabbit anti-goat Alexa-546 secondary antibody (1 : 100; Molecular Probes) was used for 1 h at room temperature, and washed three times in PBT (PBS with 1‰ Triton X-100).
Techniques: In Vitro, In Vivo, Kinase Assay, Glutathione S-Transferase Assay, Staining, Purification, Transfection, Incubation, Western Blot, Co-Immunoprecipitation Assay, Confocal Microscopy, Plasmid Preparation, Fluorescence, Expressing