rac2 protein  (Sino Biological)

Journal: Scientific Reports

Article Title: Characterization of the erythrocyte GTPase Rac1 in relation to Plasmodium falciparum invasion

doi: 10.1038/s41598-020-79052-0

Figure Lengend Snippet: Rac expression in human erythrocytes. ( A ) Proteins extracted from of 5 × 10 7 erythrocyte membranes were hybridized with a monoclonal antibody specific for Rac1 (R1-ab#2). The signal was detected at about 21 kDa, as expected. ( B ) In a different gel, same amounts of erythrocyte membranes were probed with the monoclonal anti-Rac2 specific antibody; no signal was detected. The same filter was then probed with an anti-Rac antibody (recognizing Rac1-3 proteins) (R1-ab#1), used as a loading control (bottom). The full lanes are shown in Fig. .

Article Snippet: Purified His-tagged Rac2 protein (Sino Biological) was used as a positive control.

Techniques: Expressing

Journal: bioRxiv

Article Title: PI3Kδ Bridges Microbial Surveillance with Antigen Presentation to Reinforce Intestinal Immunity

doi: 10.1101/2025.05.13.653657

Figure Lengend Snippet: ( a ) GTP-loaded RAC1/2/3 in WT, δD910A, or Nod2 - /- BM-DCs assessed by Rac G-LISA after stimulation with MDP (30 µg/ml) ( left panel) or LPS (100 ng/ml) ( right panel) (n=4-7 per group). ( b ) Representative immunoblots ( left panel) and bar chart ( right panel) showing GTP-loaded RAC2 in pulldown complexes and RAC2 in TCL from MDP-stimulated WT and δD910A BM-DCs at indicated time points. GAPDH was used to verify equal loading (n=3). ( c ) Representative immunoblots ( top and bottom, left panels) and fold change ( top and bottom , right panels) of p-AKT Ser473 levels in WT and RAC2-/- BM-DCs stimulated with MDP (30 µg/ml) or LPS (100 ng/ml) at indicated times. Middle , left, and right panels show WT BM-DCs pretreated for 2 h with EHT1864 (0.5 µM) or Veh (DMSO) before MDP stimulation (n=2-3 per group). ( d ) Representative immunoblots ( left panels) showing phagosome-associated RAC2, PI3Kδ, and NOX2 components (gp91 phox , p67 phox , p47 phox ) in WT or δD910A BM-DCs that phagocytosed 3 µM beads, coated with MDP or LPS, or left uncoated at 30 min. Phagosome-associated RAC2-GTP (magenta rectangle) was pulldown with PAK-CRIB beads from BM-DC phagosome extracts of indicated genotypes, with total RAC2 and PI3Kδ in TCL is shown (black rectangle). Ratios of active RAC2 in phagosomes ( right , upper panel) or total RAC2 ( right , lower panel) are shown (n=3). ( e ) Representative immunoblots showing recombinant His-tagged p110β and p110δ binding to recombinant GTPγS-loaded His-tagged RAC2 in a cell-free system, following immunoprecipitation of complexes using Abs directed against p110α, p110β, or p110δ. GDP-loaded His-tagged RAC2 was used as a control. Immunoblots of complexes were probed using anti-His mAb. Total recombinant protein loading visualized using anti-His mAb. One representative experiment is shown (n=2). ( f ) OT-II T-cell division after co-culture with WT or δD910A BMDCs, pretreated with RAC inhibitor (EHT-1864, 0.5 µM) or Veh (DMSO) before preloading with OVA-coated beads in the presence of LPS for 5 h (n=3 per group). ( g ) Proportions of Foxp3 + and IFN-γ + OT-II T cells after restimulation with PMA and ionomycin. Results are expressed as means ± SEM. Statistical analysis by one-way or two-way ANOVA with Tukey’s post hoc test, with p values considered * p <0.05, ** p <0.01, *** p <0.001, and **** p <0.0001.

Article Snippet: Briefly, recombinant human His-tagged RAC1 or RAC2 proteins (Cytoskeleton, Inc.) were reconstituted at 1 mg/ml in dH 2 O with 1 mM DTT.

Techniques: Western Blot, Recombinant, Binding Assay, Immunoprecipitation, Control, Co-Culture Assay

Journal: Blood

Article Title: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

doi: 10.1182/blood-2018-11-886028

Figure Lengend Snippet: RAC2 mutation occurs within the highly conserved Switch II domain. (A) Sanger sequence of RAC2 exon 3 demonstrating c.184G>A in patient 1 (*) and wild-type sequence in both parents. (B) Amino acid alignment of select members of human RHO family of GTPases. Reference sequences (RAC2 NP_002863.1, RAC1 NP_008839.2, RAC3 NP_005043.1, CDC42 NP_001782.1) are from the National Center for Biotechnology Information and aligned using Clustal W. Underline, conserved Switch I and Switch II regions; open box, E62; *Q61, D63, and Y64. (C) Three-dimensional structure of the related RAC1 (3TH5)46; blue, Switch II; pink, D57 residue; and red, E62. C, C terminus; N, N terminus.

Article Snippet: Western blot analysis was performed by standard protocols using RAC2 (Millipore), AKT, phosphorylated AKT (pAKT), and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling) primary antibodies and HRP-conjugated (Sigma Aldrich) secondary antibodies.

Techniques: Mutagenesis, Sequencing, Residue

Journal: Blood

Article Title: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

doi: 10.1182/blood-2018-11-886028

Figure Lengend Snippet: Transfection of RAC2[E62K] drives increased ROS production, increased RAC2[E62K] association with PAK-protein binding domain, increased pAKT, and increased membrane ruffling and macropinocytosis. (A) Diogenes assay to measure ROS production in COS-7 cells transfected with NADPH oxidase components (gp91phox, p47phox, p67phox) and either RAC2[WT] (top), RAC2[E62K] (middle), or GFP (bottom) without stimulation (left) or after addition of 1 μM PMA (right). (B) Cumulative ROS production without (open bar) or with (filled bar) PMA stimulation; graph shows average ± standard error of the mean (SEM) of 1 representative experiment. (C) Immunoprecipitation of COS-7 cells transfected with RAC2[WT] or RAC2[E62K] using PAK1-PBD. Graph shows average ± SEM of 3 independent experiments. ***P = .0004. (D) Lysates from COS-7 cells transfected with RAC2[WT], RAC2[E62K], or untransfected were immunoblotted and stained for total AKT (tAKT) or phospho-AKT (S473). Bands were quantified by densitometry and the ratio of active, phospho-AKT/tAKT was plotted. Graph shows average ± SEM of 3 independent experiments (supplemental Figure 2B-D). (E) Confocal images of RAW264.7 cells (top, original magnification ×333) or COS-7 cells (bottom, original magnification ×235) transfected with RAC2[WT] (left) or RAC2[E62K] (right) and GFP as a transfection control. Cells were stained with Alexa-594-phalloidin (orange) to detect F-actin and Alexa-647-anti-RAC2 (red). Images were captured by a Zeiss LSM 880 confocal microscope and processed using the ZEN 2.3 lite program.

Article Snippet: Western blot analysis was performed by standard protocols using RAC2 (Millipore), AKT, phosphorylated AKT (pAKT), and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling) primary antibodies and HRP-conjugated (Sigma Aldrich) secondary antibodies.

Techniques: Transfection, Protein Binding, Membrane, Immunoprecipitation, Staining, Control, Microscopy

Journal: Blood

Article Title: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

doi: 10.1182/blood-2018-11-886028

Figure Lengend Snippet: RAC2[E62K] has altered TIAM1-mediated GDP exchange and p50RhoGAP-mediated GTP hydrolysis. (A) GDP exchange assay using RAC2[WT] (red) and RAC2[E62K] (blue) preloaded with fluorescent mantGDP and incubated with unlabeled GDP. Intrinsic nucleotide exchange (open circles) and TIAM1-mediated exchange (filled circles) are shown. Ratio TIAM1:RAC2 = 1:1. (B) mantGDP dissociation rate of RAC2[WT] and RAC2[E62K] with and without TIAM1 calculated from panel A. Number of replicates, N = 4 for intrinsic dissociation and N = 2 for TIAM1-mediated dissociation. ***P < .0005. (P value calculated using the 1-way ANOVA followed by Tukey multiple comparison test). (C) GTP hydrolysis of RAC2[WT] and RAC2[E62K] preloaded with GTP without and with p50RhoGAP (1:500, p50RhoGAP:RAC2). Colored as in panel A. (D) GTP hydrolysis rate of RAC2[WT] and RAC2[E62K] preloaded with GTP, calculated from panel C. Data acquired in triplicate. **P < .005 (P value calculated as in panel B).

Article Snippet: Western blot analysis was performed by standard protocols using RAC2 (Millipore), AKT, phosphorylated AKT (pAKT), and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling) primary antibodies and HRP-conjugated (Sigma Aldrich) secondary antibodies.

Techniques: Incubation, Comparison

Journal: Blood

Article Title: Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects

doi: 10.1182/blood-2018-11-886028

Figure Lengend Snippet: Rac2+/E62K mice recapitulate cytopenias, superoxide production, and increased neutrophil F-actin content seen in patients. (A) Peripheral blood cells from Rac2+/+ and Rac2+/E62K mice stained for lineage markers (n = 4 each group). Top, from left: CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD20+ B cells (P value calculated using Student t test with Welch correction). (B) Splenic cell populations from Rac2+/+ and Rac2+/E62K mice identified by noted markers. Two-way ANOVA with Sidak multiple comparison test was used to determine significance. (C) Splenic T-cell subsets from Rac2+/+ and Rac2+/E62K mice identified by noted markers. Two-way ANOVA with Sidak multiple comparison test was used to determine significance. (D) Superoxide production after addition of fMLF in bone marrow neutrophils from Rac2+/+ (black lines) and Rac2+/E62K mice (red lines). Results combined from 2 independent experiments. (E) Phalloidin staining for F-actin in mouse bone marrow neutrophils from Rac2+/+ and Rac2+/E62K mice (n = 4 each group). Results combined from 2 independent experiments.

Article Snippet: Western blot analysis was performed by standard protocols using RAC2 (Millipore), AKT, phosphorylated AKT (pAKT), and glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling) primary antibodies and HRP-conjugated (Sigma Aldrich) secondary antibodies.

Techniques: Staining, Comparison

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: RAC2 expression is upregulated in ccRCC, and is associated with various clinicopathological parameters in ccRCC tissues. The mRNA expression levels of RAC2 were obtained from TCGA dataset, which contained 72 adjacent normal tissues and 534 ccRCC tissues. (A) RAC2 expression was higher in the ccRCC tissues than in the adjacent normal tissues in the 72 paired tissues from patients with ccRCC. The mRNA expression levels of RAC2 were increased in (B) ccRCC tissues than in normal tissues in TCGA, (C) Gumz renal database, (D) Lenburg renal dataset, (E) Jones renal dataset and (F) Yusenko renal dataset. The high expression of RAC2 mRNA was associated with various clinicopathological factors: (G) T stage, (H) lymph node metastasis, (I) distant metastases, (J) TNM stage and (K) G grade. **** P<0.0001; *** P<0.001; ** P<0.01. RAC2, Rac family small GTPase 2; ccRCC, clear cell renal cell carcinoma; KIRC, kidney renal clear cell carcinoma; TCGA, The Cancer Genome Atlas; TNM, Tumor-Node-Metastasis.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: Association between RAC2 mRNA expression and clinicopathological parameters of patients with clear cell renal cell carcinoma.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: High RAC2 mRNA expression is associated with poor OS in patients with ccRCC. Patient samples from The Cancer Genome Atlas were separated into two groups: Those with low RAC2 expression and those with high RAC2 expression. (A) OS of patients with ccRCC was associated with RAC2 expression. OS subanalysis in regards to RAC2 expression was conducted in subgroups of patients with ccRCC: (B) Male, (C) age ≥60 years and (D) T1 + T2 stage. RAC2, Rac family small GTPase 2; ccRCC, clear cell renal cell carcinoma; OS, overall survival.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: RAC2 expression may be a diagnostic biomarker in patients with ccRCC. (A) RAC2 effectively discriminated between ccRCC and paired normal tissues (AUC 0.9095; P<0.0001). Receiver operating characteristic curve subanalysis was performed with respect to the following subgroups of patients with ccRCC: (B) G grade, (C) T stage, (D) lymph node metastasis, (E) distant metastases, (F) Tumor-Node-Metastasis stage, (G) overal survival and (H) disease free survive. RAC2, Rac family small GTPase 2; ccRCC, clear cell renal cell carcinoma; AUC, area under the curve; OS, overall survival; DFS, disease-free survival.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Expressing, Diagnostic Assay, Biomarker Discovery

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: RAC2 was up-regulated in RCC cells and tissues. (A and B) Reverse transcription-quantitative PCR assays of RAC2 mRNA expression in normal renal tubular epithelial cells (HK-2) and renal cancer cell lines (A498, ACHN, CAKI-1, OSRC-2, 786-O), and in 50 paired tissue samples of patients with ccRCC. (C and D) Western blot assays of RAC2 expression in normal renal tubular epithelial cells (HK-2) and in renal cancer cell lines (A498, ACHN, CAKI-1, OSRC-2, 786-O) and in 50 paired tissue samples of ccRCC patients. (E) Immunohistochemistry for RAC2 expression in ccRCC tissues, adjacent normal tissues and renal angiomyolipoma tissues. The inset images are the lower magnification of the same tissue as that presented in the larger image of each set. Magnification, ×40 and ×200. RAC2 expression was normalized to β-actin expression. The values of each group are presented as the mean ± standard deviation. Bars represented the means of three independent experiments. **** P<0.0001; ** P<0.01 vs. HK-2. RAC2, Rac family small GTPase 2; ccRCC, clear cell renal cell carcinoma.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry, Standard Deviation

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: RAC2 regulates certain tumor-related pathways. Enrichment curves are shown for activated gene sets related to the (A) Toll like receptor, (B) JAK STAT and (C) P53 signaling pathways, (D) apoptosis, and the (E) cell cycle and (F) MAPK signal pathways. RAC2, Rac family small GTPase 2; NES, normalized enrichment score; FDR, false discovery rate; JAK, Janus kinases; STAT, signal transducer and activator of transcription proteins; MAPK, mitogen-activated protein kinase.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Protein-Protein interactions

Journal: International Journal of Oncology

Article Title: RAC2 acts as a prognostic biomarker and promotes the progression of clear cell renal cell carcinoma

doi: 10.3892/ijo.2019.4849

Figure Lengend Snippet: RAC2 promotes the proliferation, invasion and migration of RCC cell lines in vitro . (A and B) Reverse transcription-qauntitative PCR and western blotting assays of RAC2 knockdown in ACHN and 786-O cells; β-actin was used as a loading control. (C and D) Cell counting kit-8 assays detected the effects of RAC2 knockdown on the proliferation of ACHN and 786-O cells. (E-H) Representative images of migration and invasion assays performed using ACHN and 786-O cells (magnification, ×100). Data are presented as the mean ± standard deviation from three independent experiments. **** P<0.0001; *** P<0.001; ** P<0.01 and * P<0.05 vs. si-NC. RAC2, Rac family small GTPase 2; ccRCC, clear cell renal cell carcinoma; NC, negative control; si-RNA, small interfering RNA; OD value, optical density value.

Article Snippet: The protein expression level of RAC2 in ccRCC tissues was also obtained from The Human Protein Atlas ( http://www.proteinatlas.org ).

Techniques: Migration, In Vitro, Reverse Transcription, Western Blot, Knockdown, Control, Cell Counting, Standard Deviation, Negative Control, Small Interfering RNA

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