Total cell lysates or pull-downs were Western blotted using routine procedures. The primary antibodies used were anti-Rac (Cell Signaling), anti-Cdc42 (Abcam), anti-PAK1+PAK2+PAK3 (Cell Signaling), anti-pPAK1 (T423)/PAK2 (T402) (Abcam), Anti-PAK1 + PAK2 + PAK3 (phospho T423) (Abcam), and β -actin (Millipore-Sigma). Actin was used as a control; the Rac/Cdc42 inhibitors do not affect total actin expression, only its polymerization and organization of the cytoskeleton (23 (link)).
Protocol detail
Immunoblotting of Rho GTPases
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Corresponding Organization : University of Puerto Rico System
Other organizations : Translational Therapeutics (United States), University of Pennsylvania
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Variable analysis
independent variables
- Rac inhibitors
- Cdc42 inhibitors
- Rac expression
- Cdc42 expression
- PAK1 + PAK2 + PAK3 expression
- Phosphorylation of PAK1 (T423)/PAK2 (T402)
- Phosphorylation of PAK1 + PAK2 + PAK3 (T423)
- β-actin expression
- Positive control: β-actin
- Negative control: Not explicitly mentioned
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