sirna stam1  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Disruption of the β-arrestin1·STAM1 complex attenuates CXCR4-dependent chemotaxis. HeLa cells transiently transfected with empty vector (pCMV), FLAG-β-arrestin1(25–161), or FLAG-STAM1(296–380) were passaged onto μ-Slide chemotaxis chambers and analyzed by time lapse microscopy for 18 h in the absence (vehicle) or presence of a gradient of CXCL12 (50 nm at its source). A and B, aggregated trajectories of individual cells in the presence of vehicle (A) or CXCL12 (B) from a representative experiment. Trajectories in black and red represent cells that migrated toward or away from the chemoattractant gradient, respectively. C, the graph represents the mean forward migration index in the absence or presence of CXCL12 from tracking 150 cells from three (vehicle) or 200 cells from four (CXCL12) independent experiments. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test. p values are provided. D, aggregated trajectories of individual cells in the presence of EGF (200 ng/ml at its source) from a representative experiment. E, the forward migration index is shown from tracking 100 cells from two independent experiments. The error bars represent the S.D. Data were analyzed by Student's t test and are not significant (ns).

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Chemotaxis Assay, Transfection, Plasmid Preparation, Time-lapse Microscopy, Migration

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Depletion of β-arrestin1 or STAM1 attenuates CXCR4-dependent chemotaxis. HeLa cells transiently transfected with siRNA against β-arrestin1 or shRNA against STAM1 were passaged onto μ-Slide chemotaxis chambers and analyzed by time lapse microscopy for 18 h in the absence (vehicle) or presence of a gradient of CXCL12 (50 nm at its source). A, B, D, and E, aggregated trajectories of individual cells transfected with either βArr1 siRNA (A and B) or STAM1 shRNA (D and E) in the presence of vehicle (A and D) or CXCL12 gradient (B and E). C and F, the forward migration index was calculated from 100 control siRNA (siCtrl) (two experiments)-, 200 βArr1 siRNA (four experiments)-, 150 pLKO (three experiments)-, or 150 STAM shRNA (three experiments)-transfected cells. The error bars represent the S.D. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test. p values are provided.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Chemotaxis Assay, Transfection, shRNA, Time-lapse Microscopy, Migration

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Disruption of the β-arrestin1·STAM1 complex does not alter CXCR4 cell surface expression or CXCR4-promoted Akt or ERK-1/2 activation. A, surface expression of CXCR4 was analyzed by flow cytometry in cells transfected with empty vector (pCMV-10) or FLAG-βArr1(25–161). Cells were serum-starved for 1 h and then treated with vehicle (18 h) or 30 nm CXCL12 for 18 h, 1 h, or 5 min. Cells were fixed and stained with a phycoerythrin-conjugated antibody against CXCR4 or IgG2a (isotype control) and analyzed by flow cytometry. Bars represent the mean of the florescence intensity relative to vehicle-treated cells transfected with empty vector. The error bars represent the S.D. from two independent experiments. B and C, HeLa cells transiently transfected with FLAG-βArr1(25–161), FLAG-STAM1(296–380), or empty vector (pCMV-10) were serum-starved for 3 h and treated with 10 nm CXCL12 or vehicle (PBS with 0.1% BSA) for 5 min. Whole cell lysates were analyzed by immunoblotting for the indicated proteins. Representative immunoblots from three independent experiments are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Expressing, Activation Assay, Flow Cytometry, Transfection, Plasmid Preparation, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: RNA interference against β-arrestin1 or STAM1 attenuates CXCR4-promoted autophosphorylation of FAK. HeLa cells transfected with β-arrestin1 siRNA (A) or STAM1 shRNA (B) were treated with 10 nm CXCL12 for 5 min and analyzed as in Fig. 5A. Representative immunoblots from five (A) or seven (B) independent experiments are shown. Graphs represent the densitometric analyses showing the relative levels of Tyr(P)-397-FAK (pFAK) compared with the control (siCtrl in A or pLKO in B)-transfected cells treated with CXCL12. The error bars represent the S.D. Data were analyzed by two-way ANOVA and Newman-Keuls multiple comparison test. p values between the indicated groups are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Transfection, shRNA, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: FAK exists in a CXCR4-promoted complex with β-arrestin1 and STAM1. HeLa cells transfected with β-arrestin1-FLAG and T7-STAM1 were treated with 10 nm CXCL12 for 5, 15, 30, or 60 min and vehicle for 60 min. For controls, HeLa cells transfected with pCMV (empty vector) were treated with vehicle or 10 nm CXCL12 for 30 min. Cleared lysates were immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates and lysates were analyzed by immunoblotting for the indicated proteins. Representative immunoblots from three independent experiments are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Localization of FAK, β-arrestin1-GFP, and T7-STAM1 by fluorescence microscopy. A, HeLa cells transfected with β-arrestin1-GFP and T7-STAM1 seeded onto coverslips were treated with 10 nm CXCL12 for 5 or 30 min or for 30 min with vehicle. Images were acquired for β-arrestin1-GFP (green), FAK (red), and T7-STAM1 (blue) using identical acquisition settings for parallel samples in each channel. B, the fluorescence intensity profiles of the indicated lines (a–i) within the merged images are shown. C, colocalization analysis among the indicated proteins is shown as nMDP values for 60 ROIs (three independent experiments, five cells per experiment, four ROIs per cell). The error bars represent the S.D. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. p values are provided. Distance per pixel was calibrated to equal 0.156 μm. Scale bar, 20 μm. a.u., arbitrary units.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Fluorescence, Microscopy, Transfection

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Disruption of the β-arrestin1·STAM1 complex attenuates CXCR4-dependent chemotaxis. HeLa cells transiently transfected with empty vector (pCMV), FLAG-β-arrestin1(25–161), or FLAG-STAM1(296–380) were passaged onto μ-Slide chemotaxis chambers and analyzed by time lapse microscopy for 18 h in the absence (vehicle) or presence of a gradient of CXCL12 (50 nm at its source). A and B, aggregated trajectories of individual cells in the presence of vehicle (A) or CXCL12 (B) from a representative experiment. Trajectories in black and red represent cells that migrated toward or away from the chemoattractant gradient, respectively. C, the graph represents the mean forward migration index in the absence or presence of CXCL12 from tracking 150 cells from three (vehicle) or 200 cells from four (CXCL12) independent experiments. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test. p values are provided. D, aggregated trajectories of individual cells in the presence of EGF (200 ng/ml at its source) from a representative experiment. E, the forward migration index is shown from tracking 100 cells from two independent experiments. The error bars represent the S.D. Data were analyzed by Student's t test and are not significant (ns).

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Chemotaxis Assay, Transfection, Plasmid Preparation, Time-lapse Microscopy, Migration

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Depletion of β-arrestin1 or STAM1 attenuates CXCR4-dependent chemotaxis. HeLa cells transiently transfected with siRNA against β-arrestin1 or shRNA against STAM1 were passaged onto μ-Slide chemotaxis chambers and analyzed by time lapse microscopy for 18 h in the absence (vehicle) or presence of a gradient of CXCL12 (50 nm at its source). A, B, D, and E, aggregated trajectories of individual cells transfected with either βArr1 siRNA (A and B) or STAM1 shRNA (D and E) in the presence of vehicle (A and D) or CXCL12 gradient (B and E). C and F, the forward migration index was calculated from 100 control siRNA (siCtrl) (two experiments)-, 200 βArr1 siRNA (four experiments)-, 150 pLKO (three experiments)-, or 150 STAM shRNA (three experiments)-transfected cells. The error bars represent the S.D. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparison test. p values are provided.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Chemotaxis Assay, Transfection, shRNA, Time-lapse Microscopy, Migration

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Disruption of the β-arrestin1·STAM1 complex does not alter CXCR4 cell surface expression or CXCR4-promoted Akt or ERK-1/2 activation. A, surface expression of CXCR4 was analyzed by flow cytometry in cells transfected with empty vector (pCMV-10) or FLAG-βArr1(25–161). Cells were serum-starved for 1 h and then treated with vehicle (18 h) or 30 nm CXCL12 for 18 h, 1 h, or 5 min. Cells were fixed and stained with a phycoerythrin-conjugated antibody against CXCR4 or IgG2a (isotype control) and analyzed by flow cytometry. Bars represent the mean of the florescence intensity relative to vehicle-treated cells transfected with empty vector. The error bars represent the S.D. from two independent experiments. B and C, HeLa cells transiently transfected with FLAG-βArr1(25–161), FLAG-STAM1(296–380), or empty vector (pCMV-10) were serum-starved for 3 h and treated with 10 nm CXCL12 or vehicle (PBS with 0.1% BSA) for 5 min. Whole cell lysates were analyzed by immunoblotting for the indicated proteins. Representative immunoblots from three independent experiments are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Expressing, Activation Assay, Flow Cytometry, Transfection, Plasmid Preparation, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: RNA interference against β-arrestin1 or STAM1 attenuates CXCR4-promoted autophosphorylation of FAK. HeLa cells transfected with β-arrestin1 siRNA (A) or STAM1 shRNA (B) were treated with 10 nm CXCL12 for 5 min and analyzed as in Fig. 5A. Representative immunoblots from five (A) or seven (B) independent experiments are shown. Graphs represent the densitometric analyses showing the relative levels of Tyr(P)-397-FAK (pFAK) compared with the control (siCtrl in A or pLKO in B)-transfected cells treated with CXCL12. The error bars represent the S.D. Data were analyzed by two-way ANOVA and Newman-Keuls multiple comparison test. p values between the indicated groups are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Transfection, shRNA, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: FAK exists in a CXCR4-promoted complex with β-arrestin1 and STAM1. HeLa cells transfected with β-arrestin1-FLAG and T7-STAM1 were treated with 10 nm CXCL12 for 5, 15, 30, or 60 min and vehicle for 60 min. For controls, HeLa cells transfected with pCMV (empty vector) were treated with vehicle or 10 nm CXCL12 for 30 min. Cleared lysates were immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates and lysates were analyzed by immunoblotting for the indicated proteins. Representative immunoblots from three independent experiments are shown.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: β-Arrestin1 and Signal-transducing Adaptor Molecule 1 (STAM1) Cooperate to Promote Focal Adhesion Kinase Autophosphorylation and Chemotaxis via the Chemokine Receptor CXCR4 *

doi: 10.1074/jbc.M116.757138

Figure Lengend Snippet: Localization of FAK, β-arrestin1-GFP, and T7-STAM1 by fluorescence microscopy. A, HeLa cells transfected with β-arrestin1-GFP and T7-STAM1 seeded onto coverslips were treated with 10 nm CXCL12 for 5 or 30 min or for 30 min with vehicle. Images were acquired for β-arrestin1-GFP (green), FAK (red), and T7-STAM1 (blue) using identical acquisition settings for parallel samples in each channel. B, the fluorescence intensity profiles of the indicated lines (a–i) within the merged images are shown. C, colocalization analysis among the indicated proteins is shown as nMDP values for 60 ROIs (three independent experiments, five cells per experiment, four ROIs per cell). The error bars represent the S.D. Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. p values are provided. Distance per pixel was calibrated to equal 0.156 μm. Scale bar, 20 μm. a.u., arbitrary units.

Article Snippet: The target sequence selected for design of the short hairpin STAM1 (shSTAM1) construct was based on the following siRNA against STAM1 from GE Dharmacon (siGENOME siRNA catalog number D-011423-01-0010), which we have used previously ( 23 ): 5′-GAACGAAGAUCCGAUGUAUUC-3′.

Techniques: Fluorescence, Microscopy, Transfection