gst rbd  (Cytoskeleton Inc)

Journal: PLoS ONE

Article Title: RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

doi: 10.1371/journal.pone.0042074

Figure Lengend Snippet: Transient RhoA activation in BCEC after treatment with thrombin (2 U/ml). Freshly prepapred lysates from BCEC before thrombin exposure (0 min) and after 0.5 min, 1 min, 3 min, 5 min and 15 min after thrombin exposure were assessed. Active RhoA was isolated using GST-Rhotekin-RBD beads following the manufacturer’s protocol. In vitro GTP S-loaded RhoA was used as a positive control (square inset). Both the active RhoA levels as well as the total RhoA levels were analyzed by immunoblotting using anti-RhoA. The double lines indicate samples were taken from a different part from the same blot and exposure. (B) Quantitative analysis of 3 independent experiments, in which the activated RhoA signals were normalized to the signal of the maximal activatable RhoA obtained using GTP S treatment. Data represent mean S.E.M.

Article Snippet: Activation of Rho GTPase was examined by GST pull-down of GTP-bound Rho proteins by using the Rho-binding domain (RBD) of Rhothekin protein (an effector of RhoA) expressed as a GST-fusion protein and coupled to agarose beads (RT02, Cytoskeleton, Inc.) to capture the active (GTP-bound) Rho proteins in BCEC-lysates.

Techniques: Activation Assay, Isolation, In Vitro, Positive Control, Western Blot

Journal: PLoS ONE

Article Title: RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

doi: 10.1371/journal.pone.0042074

Figure Lengend Snippet: Western-blot analysis of RhoA activity after BCEC exposure to thrombin (0.5, 1.5 and 15 min) obtained from lysates of untreated BCEC and BCEC pretreated for 3 hours with C3 at 37°C. Active RhoA fraction was isolated from freshly prepared BCEC lysates using GST-Rhotekin-RBD beads following the manufacturer’s protocol (upper blot). In vitro GTP S-loaded RhoA was used as a positive control. The total RhoA levels were determined in all lysates (lower blot).

Article Snippet: Activation of Rho GTPase was examined by GST pull-down of GTP-bound Rho proteins by using the Rho-binding domain (RBD) of Rhothekin protein (an effector of RhoA) expressed as a GST-fusion protein and coupled to agarose beads (RT02, Cytoskeleton, Inc.) to capture the active (GTP-bound) Rho proteins in BCEC-lysates.

Techniques: Western Blot, Activity Assay, Isolation, In Vitro, Positive Control

Journal: PLoS ONE

Article Title: Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity

doi: 10.1371/journal.pone.0048344

Figure Lengend Snippet: (a) of the Matrigel invasion assay for the different colon cancer cell lines. Cells that had invaded through Matrigel were detected on the lower side of the filter by fluorescence and counted as described in . The values are the mean +/− SD (error bars) of at least three independent experiments. (b) Cells were lysed and the level of GTP-bound Cdc42 was measured as described in . Cdc42-GTP precipitated with GST-PAK1 and total Cdc42 in the lysates were detected by immunoblotting with an anti-Cdc42 antibody. Values are the mean +/− SD (error bars) of at least three independent experiments. (c) Cells were lysed and the level of GTP-bound Rac1 was measured as described in . Rac1-GTP precipitated with GST-PAK1 and total Rac1 in the lysates were detected by immunoblotting with an anti-Rac1 antibody. Values are the mean +/− SD (error bars) of at least three independent experiments. (d) Cells were lysed and the level of GTP-bound RhoA was measured as described in . RhoA-GTP precipitated with GST-RBD and total RhoA in the lysates were determined by immunoblotting with an anti-RhoA antibody. Values are the mean +/− SD (error bars) of at least three independent experiments.

Article Snippet: Briefly, 3.10 6 cells were lysed before incubation with the GST–PAK fusion protein (PAK = the Cdc42 binding domain of human PAK1B, amino acids 56–272), for Cdc42 and Rac1 activity, or with the GST-RBD fusion protein (RBD = the Rho-binding domain of human Rhotekin, amino acids 7–89), for RhoA, coupled to Glutathione–Sepharose beads (Cytoskeleton).

Techniques: Invasion Assay, Fluorescence, Western Blot