Journal: Journal of Cell Science

Article Title: RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

doi: 10.1242/jcs.140434

Figure Lengend Snippet: RhoJ interacts and colocalises with β-PIX, GIT1 and GIT2. (A) HUVECs stably expressing GFP or GFP–daRhoJ were lysed and pulldowns performed using GFP-trap beads. Samples were probed by western blot for interactions with β-PIX, GIT1 and GIT2, using GFP as a binding control. (B) Yeast two-hybrid assays were performed using yeast transformed with Gal4 activation domain (AD) fusions of candidate interacting partners and Gal4 DNA-binding domain (DBD) fusions of daRhoJ or dnRhoJ. Positive interactions are indicated by growth of yeast on plates lacking Histidine (–His). Truncations of GIT1 were performed to map its interaction with RhoJ, with the Spa homology domain (SHD) found to be necessary for this binding. (C) HUVECs transduced to express GFP–daRhoJ were fixed and stained for GIT1, GIT2 and β-PIX. The box indicates the enlarged area. Scale bar: 20 µM. These data are representative of three independent experiments.

Article Snippet: For western blotting, mouse monoclonal anti-human RhoJ (Abcam, Cambridge, UK), mouse monoclonal anti-chicken tubulin (Sigma-Aldrich, Gillingham, UK); rabbit polyclonal anti-human GIT1 (Cell Signaling Technology, Hitchin, UK); mouse monoclonal anti-GFP (Clone 3E1, Cancer Research UK, London, UK); and rabbit monoclonal anti-phospho-GIT2 (Tyr392) (Cell Signaling Technology, Hitchin, UK) antibodies were used.

Techniques: Stable Transfection, Expressing, Western Blot, Binding Assay, Control, Transformation Assay, Activation Assay, Staining

Journal: Journal of Cell Science

Article Title: RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

doi: 10.1242/jcs.140434

Figure Lengend Snippet: GFP–daRhoJ expression increases recruitment of partner proteins to focal adhesions. (A) HUVECs were transduced to express GFP or GFP–daRhoJ, and were fixed and stained for vinculin and either β-PIX, GIT1 or GIT2. Scale bar: 20 µm. (B) Cellular lysates were prepared from HUVECs expressing GFP or GFP–daRhoJ and western blotted for GIT2 phosphorylated on Y392 (pGIT2), GIT2, GFP and tubulin. (C) HUVECs were transfected with control siRNA (siControl) or RhoJ siRNA duplexes. After 48 hours, cells were lysed and blotted for GIT2 phosphorylated on Y392, GIT2, RhoJ and tubulin. These data are representative of three independent experiments.

Article Snippet: For western blotting, mouse monoclonal anti-human RhoJ (Abcam, Cambridge, UK), mouse monoclonal anti-chicken tubulin (Sigma-Aldrich, Gillingham, UK); rabbit polyclonal anti-human GIT1 (Cell Signaling Technology, Hitchin, UK); mouse monoclonal anti-GFP (Clone 3E1, Cancer Research UK, London, UK); and rabbit monoclonal anti-phospho-GIT2 (Tyr392) (Cell Signaling Technology, Hitchin, UK) antibodies were used.

Techniques: Expressing, Staining, Western Blot, Transfection, Control

Journal: Journal of Cell Science

Article Title: RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

doi: 10.1242/jcs.140434

Figure Lengend Snippet: Reciprocal regulation of the recruitment of RhoJ, GIT1/2 and β-PIX to focal adhesions. (A) HUVECs were transfected with control siRNA (siControl), RhoJ siRNA, β-PIX siRNA or both GIT1 and GIT2 (GIT1/2) siRNA duplexes. After 48 hours, HUVECs were fixed and stained for vinculin, and RhoJ, β-PIX, GIT1 or GIT2. For each experiment, the mean grey value of either GIT1, GIT2, RhoJ or β-PIX staining (as indicated) was calculated for 20 adhesions per cell from three cells using ImageJ from three independent experiments according to the Materials and Methods. For each replicate experiment, all data points were scaled to the mean of the siControl, which was set at 100. Plotted are the means of all the scaled data points from each condition from each of the experimental replicates (mean±s.e.m.). *** P <0.001 (Mann–Whitney test comparing each of the data points to the siControl). (B) Cells were lysed after 48 and 72 hours and blotted for GIT1, GIT2, RhoJ, β-PIX or tubulin as a loading control.

Article Snippet: For western blotting, mouse monoclonal anti-human RhoJ (Abcam, Cambridge, UK), mouse monoclonal anti-chicken tubulin (Sigma-Aldrich, Gillingham, UK); rabbit polyclonal anti-human GIT1 (Cell Signaling Technology, Hitchin, UK); mouse monoclonal anti-GFP (Clone 3E1, Cancer Research UK, London, UK); and rabbit monoclonal anti-phospho-GIT2 (Tyr392) (Cell Signaling Technology, Hitchin, UK) antibodies were used.

Techniques: Transfection, Control, Staining, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

doi: 10.1242/jcs.140434

Figure Lengend Snippet: Knockdown of RhoJ, β-PIX, both GIT1 and GIT2 or a combination of all four similarly impairs tube formation. (A) HUVECs were transfected with control siRNA (siControl), RhoJ siRNA, β-PIX siRNA or GIT1 and GIT2 siRNA together (GIT1/2) or a combination of RhoJ, β-PIX, GIT1 and GIT2 duplexes. At 48 hours after transfection, the cells were replated on Matrigel and imaged after 12 and 24 hours. Scale bars: 200 µm. (B) Analysis of the tubule formation using the angiogenesis analyser ImageJ plugin to show the number of loops formed by the tubules. For each experiment the mean loop number was calculated from five to six fields of view per time point. The mean of these values from three experimental replicates is plotted ( n = 3) (mean±sem). All knockdowns give a statistically significant difference compared with the siControl duplex [ P <0.05 (Student's t -test)], but there are no differences between the individual RhoJ, β-PIX, GIT1/2 knockdowns and knockdown of all four in combination.

Article Snippet: For western blotting, mouse monoclonal anti-human RhoJ (Abcam, Cambridge, UK), mouse monoclonal anti-chicken tubulin (Sigma-Aldrich, Gillingham, UK); rabbit polyclonal anti-human GIT1 (Cell Signaling Technology, Hitchin, UK); mouse monoclonal anti-GFP (Clone 3E1, Cancer Research UK, London, UK); and rabbit monoclonal anti-phospho-GIT2 (Tyr392) (Cell Signaling Technology, Hitchin, UK) antibodies were used.

Techniques: Knockdown, Transfection, Control

Journal: Journal of Cell Science

Article Title: RhoJ interacts with the GIT–PIX complex and regulates focal adhesion disassembly

doi: 10.1242/jcs.140434

Figure Lengend Snippet: A model for RhoJ function. Active RhoJ interacts with the GIT–PIX complex through its interaction with the Spa homology domain (SHD) of GIT1 or GIT2 (GIT). GIT interacts with paxillin and β-PIX. Active RhoJ together with the GIT–PIX complex promote activation of Rac and Cdc42 and focal adhesion disassembly and increased motility. This is associated with decreased RhoA activity and decreased actinomyosin contractility.

Article Snippet: For western blotting, mouse monoclonal anti-human RhoJ (Abcam, Cambridge, UK), mouse monoclonal anti-chicken tubulin (Sigma-Aldrich, Gillingham, UK); rabbit polyclonal anti-human GIT1 (Cell Signaling Technology, Hitchin, UK); mouse monoclonal anti-GFP (Clone 3E1, Cancer Research UK, London, UK); and rabbit monoclonal anti-phospho-GIT2 (Tyr392) (Cell Signaling Technology, Hitchin, UK) antibodies were used.

Techniques: Activation Assay, Activity Assay