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    Millipore gfp rufy3
    PAK1 positively regulates <t>RUFY3</t> expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The <t>GFP-RUFY3</t> and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot
    Gfp Rufy3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 positively regulates RUFY3 expression. ( a and b ) The protein level of RUFY3 was increased by the ectopic PAK1 expression level enhancing. ( a ) A dose-dependent increase of PAK1 plasmids were transfected into SGC-7901 cells (left panel) and MKN45 cells (right panel). Western blot assays were performed to detect the protein level of endogenous RUFY3. ( b )The GFP-RUFY3 and the increasing amounts of PAK1 expression vector were transfected into SGC-7901 cells, after 24 h of transfection, the protein levels of His-PAK1 and GFP-RUFY3 were measured by western blot. ( c and d ) Inhibition of PAK1 expression can also decrease RUFY3 expression. ( c ) The SGC-7901 cells expressing GFP-RUFY3 were treated with PAK1-siRNA, and the protein levels of PAK1 and GFP-RUFY3 were measured by western blot. ( d ) The stable expressing PAK1-shRNA lentivirus SGC-7901 cells (left panel) and BGC-823 (right panel) cells were treated with two different shRNAs (nos. 1 and 2) targeting RUFY3, and the protein level of endogenous PAK1 and RUFY3 were measured by western blot

    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: Expressing, Transfection, Western Blot, Plasmid Preparation, Inhibition, shRNA

    PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: PAK1 regulates RUFY3-mediated cell migration and invasion. ( a ) Overexpression of PAK1 facilitates RUFY3-induced cell invasion. SGC-7901 cells were transfected with GFP-RUFY3 and myc-PAK1 or GFP vector and myc-PAK1, and were subjected to performing transwell invasion assay. Photographs represented the cells traveling through the micropore membrane. (Left panel) Representative photomicrographs of transwell results were taken under × 200 original magnifications. Scale bars, 50 μ m. (Middle panel) Number of invading cells is shown. The number of cells was counted in 16 independent symmetrical visual fields under the microscope ( × 400 original magnification) from three independent experiments (* 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: Migration, Over Expression, Transfection, Plasmid Preparation, Transwell Invasion Assay, Microscopy

    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

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    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

    Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** P

    Journal: Cell Death & Disease

    Article Title: PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

    doi: 10.1038/cddis.2015.50

    Figure Lengend Snippet: Overexpression of RUFY3 induces the formation of F-actin-enriched protrusion at the cell periphery. ( a ) GFP-RUFY3 localizes in F-actin-enriched invadopodia at the cell periphery of SGC-7901 cells. (Left panel) The living cell image acquisition was performed at 25 °C with SGC-7901 cells transfected with GFP-RUFY3 and undergoing a scratch wound assay, and GFP vector was used as a control. A representative image was shown. The white boxed areas in the left images ( × 100; scale bars, 200 μ m) are magnified in the right images ( × 600; scale bars, 24 μ m). The red boxed area in the right images shows that the cells expressing GFP-RUFY3 can localize at the periphery in a scratch area. (Right panel) Histogram showed the relative percentage of cells with actin protrusion at the migrating edge. Data are the average of at least three independent experiments with similar results, in which ~100 cells were counted (** 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: Over Expression, Transfection, Scratch Wound Assay Assay, Plasmid Preparation, Expressing