RhoA activity was analyzed using Rhotekin Rho binding domain (Upstate Biotechnology; Thermo Fisher Scientific, Inc.) bound to glutathione agarose beads to pulldown the active GTP-bound RhoA form from ovarian cell lysates in lysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 100 mM NaCl, 0.2% deoxycholic acid, 10% glycerol, and 10 mM MgCl2) supplemented with protease and phosphatase inhibitors (37 (link)). GTP-bound RhoA and total RhoA were evaluated by western blot analysis as above, using RhoA antibody (67B9; 1:1,000; Cell Signaling Technology, Inc.).
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RhoA Activity Assay in Ovarian Cells
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Agarose
Antibody
Buffer
Deoxycholic acid
Glutathione
Glycerol
Hepes
Inhibitors
Mgcl2
Nacl
Np 40
Ovarian
Phosphatase
Protease
Rhoa
Rhotekin
Western blot analysis
Corresponding Organization :
Other organizations : Shanghai First Maternity and Infant Hospital
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Variable analysis
independent variables
- RhoA activity
- GTP-bound RhoA
- Total RhoA
- Lysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 100 mM NaCl, 0.2% deoxycholic acid, 10% glycerol, and 10 mM MgCl2)
- Protease and phosphatase inhibitors
- Positive control: Rhotekin Rho binding domain (Upstate Biotechnology; Thermo Fisher Scientific, Inc.) bound to glutathione agarose beads to pulldown the active GTP-bound RhoA form
- Negative control: Not explicitly mentioned
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