Rhoa activation assay kit

Manufactured by Cytoskeleton

Sourced in United States

The RhoA activation assay kit is a tool designed to measure the activation state of the RhoA GTPase protein. It provides a direct and quantitative method for determining the amount of active, GTP-bound RhoA in cell and tissue samples. The kit includes necessary reagents and protocols to perform the assay.

Automatically generated - may contain errors

Lab products found in correlation

Poly hema, by Merck Group (1 mentions) Rac1 activation assay kit, by Cytoskeleton (1 mentions) Rac1 antibody, by Merck Group (1 mentions) G lisa rhoa activation assay biochem kit, by Cytoskeleton (1 mentions) Integrin αv, by Santa Cruz Biotechnology (1 mentions) Integrin α5, by Santa Cruz Biotechnology (1 mentions) P fak, by Santa Cruz Biotechnology (1 mentions)

13 protocols using rhoa activation assay kit

Quantifying RhoA Activity in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

A RhoA activity assay was performed and quantified using the RhoA activation assay kit according to the manufacturer's instructions (Cytoskeleton, Denver, CO, USA). Cells were treated with 20 nM siRNA before use and were seeded in 5 mg/ml polyHEMA (poly 2-hydroxyethyl methacrylate; Sigma)-coated 100-mm dishes for cultivation under anchorage-independent conditions overnight (Fukazawa et al., 1995 (link)). Cells were stimulated with 100 ng/ml VEGF-A₁₆₅ for 10 min or the indicated time. Cells were lysed with Lysis buffer. The clarified cell lysate was incubated with Rhotekin-RBD protein agarose beads and rotated at 4°C for 90 min. The beads were washed with wash buffer and denatured. Active RhoA was detected by western blotting.

Yoshida A., Shimizu A., Asano H., Kadonosono T., Kondoh S.K., Geretti E., Mammoto A., Klagsbrun M, & Seo M.K. (2015). VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells. Biology Open, 4(9), 1063-1076.

+ Open protocol

+ Expand

Rac1 and RhoA Activity Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell culture dishes were coated with fibronectin at 5 μg/ml or Fbln7-C at 20 μg/ml overnight at 4°C. HUVECs were plated and allowed to attach for 2 hours. Rac1 activity was assayed using the Rac1 Activation Assay Kit from Cytoskeleton Inc. (USA). Cell lysates were incubated with PAK-PBD agarose beads at 4°C for 1 hour. After washing, the proteins attached to the beads were solubilized by boiling in a LDS-sample buffer with β-mercaptoethanol. SDS-polyacrylamide gel electrophoreses (PAGE) was performed, and the proteins were detected by immunoblotting using a Rac1 antibody (Merck, IRL). RhoA activity was assayed using the RhoA Activation Assay Kit from Cytoskeleton. Cell lysates were incubated with rhotekin RBD agarose beads at 4°C for 1 hour. After washing, the proteins attached to the beads were solubilized by boiling in a LDS-sample buffer with β-mercaptoethanol. SDS-PAGEs were performed, and the proteins were detected by immunoblotting using a RhoA antibody (Abcam, USA).

de Vega S., Suzuki N., Nonaka R., Sasaki T., Forcinito P., Arikawa-Hirasawa E, & Yamada Y. (2014). A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture. Archives of biochemistry and biophysics, 545, 148-153.

+ Open protocol

+ Expand

Measuring RhoA Activity via Activation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

RhoA activity was measured using a RhoA Activation Assay kit (Cytoskeleton, Denver, CO) according to the manufacturer’s instructions. Briefly, a cell lysate was incubated at 4 °C for 1 h with a GST fusion protein containing the Rho-binding domain of rhotekin (GST-RBD) immobilized on glutathione-Sepharose beads. After washing, the bead-bound proteins were fractionated by SDS‒PAGE and immunoblotted with an anti-RhoA antibody. The total cell lysate was also blotted with the anti-RhoA antibody to permit assessment of the fractional ratio of RhoA binding protein.

Zhang T., Hu R., Wang Y., Guo S., Wu Z., Liu J., Han C., Qiu C, & Deng G. (2023). Extracellular matrix stiffness mediates uterine repair via the Rap1a/ARHGAP35/RhoA/F-actin/YAP axis. Cell Communication and Signaling : CCS, 21, 22.

+ Open protocol

+ Expand

RhoA Activation Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

RhoA activity was measured by a pull-down assay according to the manufacturer’s instructions for a RhoA Activation Assay Kit (Cytoskeleton, Denver, USA). Cells were detached from the plates by scraping with a cell scraper and solubilized on ice for 20 min in lysis buffer (125 mM HEPES, pH 7.5, 750 mM NaCl, 5% NP-40, 50 mM MgCl₂, 5 mM EDTA, 10% Glycerol). GTP-bound RhoA was then immunoprecipitated from lysate with Rho-binding domain (RBD) of Rhotekin bound to agarose beads. The beads were washed and RhoA was analyzed by Western blot analysis using RhoA-specific monoclonal antibody. The lysate (20 μg) was also probed for RhoA and β-actin to quantify protein.

Suchanski J., Tejchman A., Zacharski M., Piotrowska A., Grzegrzolka J., Chodaczek G., Nowinska K., Rys J., Dziegiel P., Kieda C, & Ugorski M. (2017). Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression. PLoS ONE, 12(9), e0184970.

+ Open protocol

+ Expand

Quantifying Active RhoA Levels

Check if the same lab product or an alternative is used in the 5 most similar protocols

GTP-bound RhoA was detected using a RhoA activation assay kit (Cytoskeleton, BK036) according to the manufacturer's protocol. Briefly, mIMCD3 cells were lysed and centrifuged. A fraction of the supernatant was saved for analysis of total RhoA, and the rest was incubated with rhotekin's Rho-binding domain-conjugated beads for 1 h at 4°C on a rocker. The beads were washed, precipitated, and subjected to Western blot analysis.

Cai J., Song X., Wang W., Watnick T., Pei Y., Qian F, & Pan D. (2018). A RhoA–YAP–c-Myc signaling axis promotes the development of polycystic kidney disease. Genes & Development, 32(11-12), 781-793.

+ Open protocol

+ Expand

RhoA Activation Assay in Lung Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

GTP-bound RhoA protein measurement was carried out using RhoA activation assay kit (Cytoskeleton, Inc., USA). Briefly, lung tissues were fragmented and lysed in 1 × ice-cold assay/lysis buffer, and then centrifuged. Each sample's supernatant was fixed with 40 μl of either rhotekin RBD or PAK PBD agarose bead slurry, and the mixture was incubated at 4 °C for 1 h with slow agitation. Afterwards, the agarose beads were centrifuged, pelleted, and then resuspended after washed for 3 times. The precipitated GTP-RhoA expression was detected by western blot using anti-RhoA antibody.

Lin X., Wang L., Lu X., Zhang Y., Zheng R., Chen R, & Zhang W. (2023). Targeting of G-protein coupled receptor 40 alleviates airway hyperresponsiveness through RhoA/ROCK1 signaling pathway in obese asthmatic mice. Respiratory Research, 24, 56.

+ Open protocol

+ Expand

Measuring RhoA Activation in Cancer

Check if the same lab product or an alternative is used in the 5 most similar protocols

To determine RhoA activation in cancer cells co-cultured with CAFs, active RhoA-GTP was precipitated with Rhotekin-Rho-binding domain (RBD) glutathione affinity beads, using a RhoA Activation Assay Kit (Cytoskeleton) according to the manufacturer instructions. In tumor samples, RhoA activation was measured in tumor lysates with G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton).

Daubriac J., Han S., Grahovac J., Smith E., Hosein A., Buchanan M., Basik M, & Boucher Y. (2017). The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1. Oncotarget, 9(12), 10375-10387.

+ Open protocol

+ Expand

RhoA Activity Measurement via Pull-Down Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

RhoA activity was assessed via a pull-down assay using the RhoA Activation Assay Kit (Cytoskeleton Inc. Denver, CO, United States), according to the manufacturer’s guidance. Briefly, after stimulation, PMVECs were lysed with cell lysis buffer (50 mM Tris pH 7.5, 10 mM MgCl₂, 0.5 M NaCl, and 2% (vol/vol) Igepal), and GTP-bound RhoA (RhoA-GTP) was captured when the lysates were incubated with Rhotekin RBD beads (1 h, 4°C). The beads were washed four times with wash buffer (40 mM NaCl, 30 mM MgCl₂, and 25 mM Tris pH 7.5) and heated to 95°C for 2 min with 20 µl of 2x Laemmli sample buffer (125 mM Tris pH 6.8, 20% (vol/vol) glycerol, 4% (wt/vol) SDS, 0.005% (wt/vol) bromophenol blue, 5% (vol/vol) beta-mercaptoethanol), and then loaded on a 10% (vol/vol) SDS-PAGE. The expression level of RhoA-GTP was caught by Western blotting using a monoclonal antibody against RhoA. In cell lysates, the total amount of RhoA was used as an endogenous control.

Zhou Q., Jiang J., Chen G., Qian C, & Sun G. (2021). Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability. Frontiers in Pharmacology, 12, 676817.

+ Open protocol

+ Expand

Evaluating RhoA Protein Activation

Check if the same lab product or an alternative is used in the 5 most similar protocols

The amount of the GTP-bound form of the RHOA protein was evaluated with a RhoA Activation Assay kit (Cytoskeleton) (Denver, CO) according to the manufacturer's instructions. Briefly, a cell lysate was incubated at 4℃ for one h with a GST fusion protein containing the RHO-binding domain of rhotekin (GST-RBD) immobilized on glutathione-Sepharose beads. After washing, the bead-bound proteins were fractionated with SDS-PAGE and immunoblotted with an anti-RHOA antibody. The total cell lysate was also blotted with the anti-RHOA antibody to assess the fractional ratio of rhotekin-bound RHOA protein.

Kumagai S., Togashi Y., Sakai C., Kawazoe A., Kawazu M., Ueno T., Sato E., Kuwata T., Kinoshita T., Yamamoto M., Nomura S., Tsukamoto T., Mano H., Shitara K, & Nishikawa H. (2020). An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T Cells. Immunity, 53(1).

+ Open protocol

+ Expand

RhoA Activation Assay for VSMCs

Check if the same lab product or an alternative is used in the 5 most similar protocols

The RhoA activation assay was performed using a RhoA activation assay kit (Cytoskeleton) according to the manufacturer’s recommendations. Briefly, VSMCs were stretched as discussed before at 15% cyclic elongation for 15 minutes following 24 hours of serum starvation. From the total cell lysates, the active form of RhoA was precipitated using Rhotekin-RBD beads followed by boiling with sample buffer. The resultant solution was analyzed by SDS-PAGE using the aforementioned mouse anti-RhoA antibody. A His-tagged control RhoA protein was used along with a GTPγS-positive control for each experiment.

Ghosh S., Kollar B., Nahar T., Suresh Babu S., Wojtowicz A., Sticht C., Gretz N., Wagner A.H., Korff T, & Hecker M. (2015). Loss of the Mechanotransducer Zyxin Promotes a Synthetic Phenotype of Vascular Smooth Muscle Cells. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 4(6), e001712.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!