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-promoted autophosphorylation of FAK. A, HeLa cells transfected with the indicated DNA constructs were treated with CXCL12 for 5 min, and cell lysates were analyzed by immunoblotting for FAK or Tyr(P)-397-FAK. Representative immunoblots from four independent experiments are shown. Bottom, the graph represents the densitometric analyses showing the relative levels of Tyr(P)-397-FAK (pFAK) compared with the control pCMV-transfected cells treated with CXCL12 of four independent experiments. 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: A , immunostaining of HeLa cells with an anti-Tyr(P)-397-FAK (BD Biosciences) antibody.

Techniques: Transfection, Construct, 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: Validation of the Tyr(P)-397-FAK antibody for fluorescence microscopy. A, immunostaining of HeLa cells with an anti-Tyr(P)-397-FAK (BD Biosciences) antibody. HeLa cells grown on PLL-coated coverslips in serum-containing medium were treated with vehicle (DMSO) and FAK inhibitors PF573228 (PF288) (1 μm) and PF562271 (PF271) (1 μm) for 3 h. Shown are representative images. The white box indicates ROI, shown enlarged in the inset below, and is inverted. B, box plot of Tyr(P)-397-FAK intensity. The fluorescence intensity of Tyr(P)-397-FAK was determined from cells in 10 fields of view, and three to five regions of interests per field of view were analyzed. The fluorescence intensity per area of region of interest was calculated, and values are represented in a box plot. The upper limit of the box represents the 75th percentile, and the lower limit represents the 25th percentile. The horizontal line within the box represents the median. The whiskers represent the maxima and minima values. Values that exceeded two standard deviations from the mean were excluded from the analysis. p values from one-way ANOVA followed by post hoc Tukey's test are provided. C, representative images of anti-Tyr(P)-397-FAK immunostaining of HeLa cells transiently transfected with YFP-β-arrestin1(25–161) and treated with CXCL12 for 5 or 30 min and vehicle for 30 min. The arrows point to high Tyr(P)-397-FAK levels in cells stimulated with CXCL12 for 5 or 30 min. In vehicle-treated cells, arrows point to edges of cells. The asterisks mark cells that are transfected with YFP-β-arrestin1(25–161). Shown are the inverted images of YFP-β-arrestin1(25–161). D, the fluorescence intensity of Tyr(P)-397-FAK in YFP-β-arrestin1(25–161)-transfected and untransfected cells was determined from cells in 20–30 fields of view and 37 regions of interest per condition from two independent experiments. The fluorescence intensity per surface area of region of interest was calculated, and values are represented in a box plot. The upper limit of the box represents the 75th percentile, and the lower limit represents the 25th percentile. The horizontal line within the box represents the median. The whiskers represent the maxima and minima values. Values that exceeded two standard deviations from the mean were excluded from the analysis. Adjusted p values from one-way ANOVA followed by post hoc Tukey's test are provided. V, vehicle. 5 and 30 represent the time in minutes cells were treated with 10 nm CXCL12. ns, not significant.

Article Snippet: A , immunostaining of HeLa cells with an anti-Tyr(P)-397-FAK (BD Biosciences) antibody.

Techniques: Fluorescence, Microscopy, Immunostaining, 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: 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: A , immunostaining of HeLa cells with an anti-Tyr(P)-397-FAK (BD Biosciences) antibody.

Techniques: Transfection, shRNA, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Rab13-dependent Trafficking of RhoA Is Required for Directional Migration and Angiogenesis *

doi: 10.1074/jbc.M111.245209

Figure Lengend Snippet: Targeting of Syx to Tyr(P)1175-VEGFR2 by Grb2. A, Syx localization was not polarized in quiescent ECs, and the Tyr(P)1175-VEGFR2 signal at the cell edges was low. In ECs subjected to a VEGF-A164 gradient (0–20 ng/m), Syx colocalized with Tyr(P)1175-VEGFR2 (pY-R2) at the leading edge. B, quantification of the extent and angular distribution of Syx and Tyr(P)1175-VEGFR2 colocalization in ECs subjected to a VEGF-A164 gradient (0–20 ng/m). The gradient direction is 0 degrees (mean ± S.D., n = 10, p < 0.0001). C, Syx, Rab13, and Grb2 were absent from the leading edge of untreated spontaneously polarized ECs. A VEGF-A164 gradient (0–20 ng/m) induced colocalization of the three proteins in frontal lamellipodia (arrowheads). D, quantification of the colocalization of Grb2 with Rab13 within a distance of ∼5 μm from the leading edge of untreated ECs and of ECs subjected to a 0–20 ng/m VEGF-A164 gradient (mean ± S.D., n = 10, p < 3 × 10−7). E, the extent of Rab13 coimmunoprecipitated with Grb2 (molecular mass = 25.2 kDa) was higher in ECs treated by VEGF-A164 (20 ng/ml, 5 min). Scale bars, 10 μm.

Article Snippet: Antibodies to Rab13 (Millipore or Santa Cruz Biotechnology), RhoA (Abcam), Grb2 (Santa Cruz Biotechnology), Tyr(P) 1175 VEGFR2 (GenScript), and phalloidin-Alexa647 (Invitrogen) were used according to the manufacturer's instructions.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Rab13-dependent Trafficking of RhoA Is Required for Directional Migration and Angiogenesis *

doi: 10.1074/jbc.M111.245209

Figure Lengend Snippet: Hypothetical mechanism of Syx and RhoA trafficking from cell junctions to the leading edge. In step 1, VEGF-A binding to VEGFR2 triggers clathrin-dependent endocytosis of junctional transmembrane proteins. Amot targets the Syx-associated complex, containing also Mupp1 and RhoA, to the cytoplasmic leaflet of uncoated endocytic vesicles enclosing the endocytosed junctional transmembrane proteins (step 2). Rab13 associates with and mediates the trafficking of these vesicles. It recruits Grb2 (step 3), which targets the Rab13-associated vesicles to Tyr-phosphorylated VEGFR2. Syx activates RhoA cotrafficking on the same vesicles, at the leading edge of the migrating cell.

Article Snippet: Antibodies to Rab13 (Millipore or Santa Cruz Biotechnology), RhoA (Abcam), Grb2 (Santa Cruz Biotechnology), Tyr(P) 1175 VEGFR2 (GenScript), and phalloidin-Alexa647 (Invitrogen) were used according to the manufacturer's instructions.

Techniques: Binding Assay