Materials
Name | Company | Catalog Number | Comments |
Bugbuster | Novagen | 70584-3 | |
COMPLETE protease inhibitor | Roche | 05 056 489 001 | |
Glutathione magnetic beads | Pierce | 88821 | |
Polyethylenimine, branched, average Mw ~25,000 | Sigma Aldrich | 408727-100ML | |
OPIMEM | Life Technologies | 31985-047 | |
Dulbecco's Modified Eagle Media | Sigma Aldrich | D5796 | |
Fetal Bovine Serum | Life Technologies | 10270-1-6 | |
L-Glutamine | Life Technologies | 25030-024 | |
GFP-Trap_A | Chromotec | gta-20 | |
GDP | Sigma Aldrich | G7127 | Highly unstable. Aliquot and store at -80 immediately upon reconstritution |
GTPγS | Sigma Aldrich | G8634 | Highly unstable. Aliquot and store at -80 immediately upon reconstritution |
Blocking Buffer | Sigma Aldrich | B6429 | |
Tween-20 | Sigma Aldrich | P9416 | |
Anti-GFP antibody | Living Colors | 632592 | Use at 1/1000 dilution |
DyLight 800 conjugated goat anti-rabbit secondary antibody | Fisher Scientific | 10733944 | |
Anti-PAK1 antibody | Cell Signaling | 2602S | Use at 1/1000 dilution |
Odyssey SA Infrared Imaging System | Li-cor | 9260-11PC |
References
- Burridge, K., Rho Wennerberg, K. and Rac take center stage. Cell. 116 (2), 167-179 (2004).
- Raftopoulou, M., Hall, A. Cell migration: Rho GTPases lead the way. Dev Biol. 265 (1), 23-32 (2004).
- Rossman, K. L., Der, C. J., Sondek, J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).
- Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature. 408 (6813), 682-688 (2000).
- Scheffzek, K., Ahmadian, M. R. GTPase activating proteins: structural and functional insights 18 years after discovery. Cell Mol Life Sci. 62 (24), 3014-3038 (2005).
- Del Pozo,, A, M., et al. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat Cell Biol. 4 (3), 232-239 (2002).
- Williamson, R. C., et al. Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure. J Cell Sci. 127 (Pt 19), 4292-4307 (2014).
- Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. Embo J. 19 (9), 2008-2014 (2000).
- Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion. PLoS. 7 (1), e29912 (2012).
- Self, A. J., Hall, A. Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates. Methods Enzymol. 256, 67-76 (1995).