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

Journal: Haematologica

Article Title: A gain-of-function RAC2 mutation is associated with bone marrow hypoplasia and an autosomal dominant form of severe combined immunodeficiency

doi: 10.3324/haematol.2019.230250

Figure Lengend Snippet: Identification and effects of the p.G12R RAC2 mutation on the GTPase activity of RAC2. (A) Pedigrees of the three patients from two unrelated kindred. Black boxes and black circles respectively represent the affected male (P1) and affected females (P2, P3). White boxes and circles respectively represent unaffected males and females. Arrows represent the probands and a double horizontal bar represents consanguinity. (B) A representative electropherogram of RAC2 DNA sequencing for control cells and patient cells, showing the c.34G>A mutation. (C) A representative immunoblot of RAC2 protein expression in lysates from control fibroblasts (Ctrl) and fibroblasts derived from the affected individuals (P1, P2, and P3). The loading control corresponds to GAPDH expression. (D) 3D models of the RAC2 G12R mutant. The structures are shown with GDP (left panel) or GTP (right panel) in the G1 binding pocket. For each model, a close-up view of the GDP/GTP binding pocket (dotted brown circle) is shown below the overall view. The figure was generated with the pymol program ( www.pymol.org/ ). (E) HEK293T cells were either not transduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutated form described here (G12R) or (as a positive control) the constitutively activated GTP-bound RAC2 form (G12V). Two days after transduction, cells were recovered for analysis using the G-LISA assay (15 μg of total protein per well) for the quantification of the GTP-bound RAC2 form (RAC2 GTP). The results come from three independent experiments, and the table below the graph represents the mean of the percentage of GFP expressing cells (GFP + ) in the three independent experiments. *** P <0.001; **** P <0.0001.

Article Snippet: Levels of activated RAC2 were determined using the G-LISA ® RAC Activation Assay Biochem KitTM (#BK125, Cytoskeleton Inc.) according to the manufacturer’s instructions, except that RAC2 monoclonal specific antibody (AT2G11, sc-517424 Santa Cruz Biotechnology, Inc.) and HRPconjugated anti-mouse antibody (#1721011, Bio-Rad) were used to detect the amount of captured active RAC2 (details in the Online Supplementary Methods ).

Techniques: Mutagenesis, Activity Assay, DNA Sequencing, Control, Western Blot, Expressing, Derivative Assay, Binding Assay, Generated, Transduction, Plasmid Preparation, Positive Control

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: In accordance with RNA-Seq data, mRNA expression of RAC2 and PTGDS by quantitative real-time PCR was upregulated in LNs compared with primary cancers. LN indicates lymph node metastasis; N, normal breast; PCR, polymerase chain reaction; T, cancer.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: RNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Representative immunostaining patterns of RAC2 and PTGDS in normal epithelium of the breast (A, D), corresponding carcinoma (B, E), and metastatic lymph node (C, F) (A–C, RAC2; D–F, PTGDS) (×400, scale bars; 60 μm).

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: Immunostaining

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Distribution of Allred scores for RAC2 and PTGDS in normal breast tissues, corresponding carcinoma, and LN. The ends of the box represent the 25th and 75th percentiles; the bars indicate the 10th and 90th percentiles, and a horizontal line inside the box shows the median. LN indicates lymph node metastasis; N, normal breast; T, cancer.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques:

Journal: Applied Immunohistochemistry & Molecular Morphology

Article Title: Differentially Expressed Genes in Matched Normal, Cancer, and Lymph Node Metastases Predict Clinical Outcomes in Patients With Breast Cancer

doi: 10.1097/PAI.0000000000000717

Figure Lengend Snippet: Overall survival analysis based on RAC2 expression.

Article Snippet: The following probes of TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used: Hs00427439_g1 ( RAC2 ), Hs00168748_m1 ( PTGDS ), and Hs02758991_g1 (glyceraldehyde 3-phosphate dehydrogenase, GAPDH ).

Techniques: Expressing

Journal: eLife

Article Title: A novel Drosophila injury model reveals severed axons are cleared through a Draper/MMP-1 signaling cascade

doi: 10.7554/eLife.23611

Figure Lengend Snippet: Annokey analysis results of upregulated genes associated with glial membrane expansion and movement. (a) List of terms used in Annokey analysis. (b) Top ten hits associated with our Annokey key terms in Drosophila , mouse, and human. MMP-1/MMP-14 (red bold) is included in the top ten list for each species. DOI: http://dx.doi.org/10.7554/eLife.23611.011 10.7554/eLife.23611.012 Table 1—source data 1. Human HTML Annokey search results that include hyperlinks to NCBI Gene, GeneRIF and Pubmed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.012 10.7554/eLife.23611.013 Table 1—source code 1. Drosophila HTML Annokey results, including hyperlinks to NCBI gene, GeneRIF, and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.013 10.7554/eLife.23611.014 Table 1—source code 2. Mouse HTML Annokey results, including hyperlinks to NCBI Gene, GeneRIF,pu and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.014 10.7554/eLife.23611.015 Table 1—source code 3. Human HTML Annokey results, including hyperlinks to NCBI Gene, GeneRIF, and PubMed databases. DOI: http://dx.doi.org/10.7554/eLife.23611.015

Article Snippet: Additional TaqMan gene expression assays were utilized: Ets21c-Dm01814139_m1; PGRP-SA-Dm01837990_g1; MMP-1-Dm01820359_m1; Relish-Dm02134843_g1; Ninjurin A-Dm01798347_g1; Hairy- Dm01822363_m1; Rac2-Dm01840631_s1; Cactus-Dm01807760_m1; Notch-Dm01841974_g1; CG6277 – Dm02369365_s1.

Techniques: Membrane