rhoa levels  (Cytoskeleton Inc)

Journal: PLoS ONE

Article Title: Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

doi: 10.1371/journal.pone.0023736

Figure Lengend Snippet: Rat-2 fibroblasts were treated with Msp in a time course. At the indicated times, cells were lysed and the level of active Rac1 (A), RhoA (B) or Cdc42 (C) were measured using the appropriate G-LISA. The amount of active Ras (D) in cell lysates was measured using a Raf-RBD pulldown assay. Cells were treated with EGF or LPA as a positive control. Graphs represent the fold change relative to non-treated cells (mean ± SEM, * P <0.05, ** P <0.01, *** P <0.001).

Article Snippet: Following Msp treatment, lysates were prepared and Rac1 and RhoA activation levels were measured using the appropriate G-LISA as above (Cytoskeleton).

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

doi: 10.1371/journal.pone.0023736

Figure Lengend Snippet: Immunofluorescence microscopy images of fibroblasts following exposure to Msp. Rac1 (A, arrows) is localized to actin-rich areas (A, arrowheads) at the cell periphery. Msp resulted in changed distribution of both RhoA (B) and Cdc42 (C), however neither localized to areas of remodelled actin. Alexa-phalloidin was used to label F-actin while specific antibodies were used to label each small GTPase. Images are representative of 3 experiments. Bar represents 25 µm.

Article Snippet: Following Msp treatment, lysates were prepared and Rac1 and RhoA activation levels were measured using the appropriate G-LISA as above (Cytoskeleton).

Techniques: Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

doi: 10.1371/journal.pone.0023736

Figure Lengend Snippet: Prior to Msp treatment for 15 min, fibroblasts were treated with nothing, vehicle or the PI3K inhibitor LY294002 for 30 min. Activation of Rac1 and RhoA was measured in cell lysates using a G-LISA. Pretreatment with LY294002 was able to inhibit Rac1 activation (A) but not RhoA activation (B) by Msp, compared to Msp treatment alone. Graphs are representative of 3 independent experiments (mean ± SEM, * P <0.05, ** P <0.01).

Article Snippet: Following Msp treatment, lysates were prepared and Rac1 and RhoA activation levels were measured using the appropriate G-LISA as above (Cytoskeleton).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

doi: 10.1371/journal.pone.0023736

Figure Lengend Snippet: Msp treatment of fibroblasts results in activation of both PI3K and a host 3-phosphoinositide phosphatase, resulting in turnover of PIP3 to PIP2. PIP2 then causes removal of actin capping proteins, FBE formation and dependent actin filament assembly, even though cofilin remains inactive. Although Msp activates Rac1, RhoA and Ras, our data suggest that these small GTPases are not directly involved in Msp-induced subcortical actin assembly. indicates pathway activated, indicates pathway inhibited.

Article Snippet: Following Msp treatment, lysates were prepared and Rac1 and RhoA activation levels were measured using the appropriate G-LISA as above (Cytoskeleton).

Techniques: Activation Assay