Journal: Biomedical Reports

Article Title: RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2

doi: 10.3892/br.2018.1119

Figure Lengend Snippet: Comparisons of GAP activity between small G-proteins. GTP hydrolysis was determined by measuring the amount of released γPi. Following reaction, the γPi level was measured in the presence of CytoPhos reagent from absorbance at 650 nm. Hydrolysis of GTP by the indicated small G-proteins was performed either in the presence or absence of (A) GST-RASAL3 and (B) GST-p50 rhoGAP at 37°C for 15 min. The CytoPhos reagent was added to the reactions and incubated for a further 10 min at room temperature, after which the absorbance at 650 nm was measured. Means ± standard deviation of three independent experiments are represented. GAP, GTPase activating protein; GST, glutathione S transferase; RASAL3, Ras activating protein-like 3.

Article Snippet: RhoGAP Assay Biochem kit (cat. no. BK105) containing H-Ras, RhoA, Rac1 and Cdc42, separate His-Rac2 protein (cat. no. RC02) and CytoPhos Reagent were purchased from Cytoskeleton, Inc. (Denver, CO, USA).

Techniques: Activity Assay, Incubation, Standard Deviation

Journal: PLoS Pathogens

Article Title: The Origin of Intraspecific Variation of Virulence in an Eukaryotic Immune Suppressive Parasite

doi: 10.1371/journal.ppat.1001206

Figure Lengend Snippet: ( A ) Dot blot experiments on serial dilutions of the recombinant proteins GST-LbGAP and GST-LbGAPy using a LbGAP-specific polyclonal antibody. GST-tag alone was used as a control. ( B ) Slot blot on serial dilution of protein extract starting from twenty ISm and twenty ISy venom apparatus. ( C ) Slot blot on serial dilution of protein extract starting from five ISm and five F1 hybrid venom apparatus.

Article Snippet: In vitro GAP assays with recombinant GST-LbGAP and GST-LbGAPy proteins were performed in triplicates using the RhoGAP Assay Biochem Kit from Cytoskeleton Inc. P values were generated by ANOVA followed by pairwise comparisons using pairwise t tests performed with the R software package ( http://www.r-project.org/ ).

Techniques: Dot Blot, Recombinant, Serial Dilution

Journal: Biomedical Reports

Article Title: RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2

doi: 10.3892/br.2018.1119

Figure Lengend Snippet: GAP assay using full-length RASAL3 protein. The GTPase activating activity of Rac1 and Rac2 was examined in the presence or absence of whole RASAL3 protein, which was prepared using the pFLAG-CMV/hRASAL3 vector. Means ± standard deviation of three independent experiments are represented. The panel within the graph represents an image of western immunoblotting with anti-FLAG antibody (MW was determined as 120 kDa). RASAL3, Ras activating protein-like 3; GAP, GTPase activating protein; MW, molecular weight; WCL, whole cell lysate.

Article Snippet: RhoGAP Assay Biochem kit (cat. no. BK105) containing H-Ras, RhoA, Rac1 and Cdc42, separate His-Rac2 protein (cat. no. RC02) and CytoPhos Reagent were purchased from Cytoskeleton, Inc. (Denver, CO, USA).

Techniques: GAP Assay, Activity Assay, Plasmid Preparation, Standard Deviation, Western Blot, Molecular Weight

Journal: Biomedical Reports

Article Title: RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2

doi: 10.3892/br.2018.1119

Figure Lengend Snippet: In vitro binding assay between RASAL3 GAP domain and Rac2. (A) Purified His-Rac2 proteins (2 µg each) and slurries of GST-RASAL3-GAP beads (50 µl) were mixed with 50 µl 1× GAP assay reaction buffer in the presence of 20 µM GDP, GTP or GTPγS at room temperature for 30 min. The bound proteins were washed and resolved on SDS-PAGE and stained with Coomassie Blue. (B) In vitro bindings between the GST-RASAL3-GAP and each small G-protein were independently performed with 50 mM Tris-Cl (pH 7.5) containing 150 mM NaCl and 20 µM GTP at room temperature for 30 min (upper). The quantities of GTPase pulled down with GST-RASAL3 GAP domain were expressed by relative image density, which was normalized against each GTPase alone (lower). RASAL3, Ras activating protein-like 3; GAP, GTPase activating protein; GST, glutathione S transferase.

Article Snippet: RhoGAP Assay Biochem kit (cat. no. BK105) containing H-Ras, RhoA, Rac1 and Cdc42, separate His-Rac2 protein (cat. no. RC02) and CytoPhos Reagent were purchased from Cytoskeleton, Inc. (Denver, CO, USA).

Techniques: In Vitro, Binding Assay, Purification, GAP Assay, SDS Page, Staining