ANTIBODIES TO GTPase REGULATORS

When a cell receives extracellular signals it may need to activate or turn on a number of downstream signaling pathways in order to initiate a particular cellular response. The downstream signals activated by growth factors, cytokines, hormones, cell-cell interactions, or physical stress can be complex and result in the modification of a number of cellular processes. These far reaching and complex effects can be achieved via a simple biochemical concept that involves proteins that function as pivotal molecular switches and interact with a multitude of effectors to turn on or turn off particular sets of responses.

The molecular switches are G-proteins, GTPases that hydrolyze GTP and cycle between two conformational states depending on whether GTP (guanine triphosphate) or GDP (guanine diphosphate) is bound. The GTP-bound GTPase is active, while the GDP-bound is inactive. In its active GTP-bound state, GTPases recognize and interact with target effectors that propagate downstream signals. Regulation of GTPase activity is achieved via activators called GEFs (guanine-nucleotide exchange factors) and inactivators called GAPs (GTPase-activating proteins). GEFs transduce signals from receptor tyrosine kinases, G-protein-coupled receptors, adhesion molecules, and second messengers and promote GTP binding to GTPases. GTP binding and signaling to target effectors is in turn terminated by the hydrolysis of GTP to GDP facilitated by GAPs. A third class of GTPase regulators, the GDIs (G-nucleotide dissociation inhibitors), also exists. GDIs are specific regulators of the Rho/Rac and Rab families of monomeric GTPases. GDIs influence G-protein signaling by binding the GDP or GTP form of the GTPase and preventing dissociation of the nucleotide.

It is apparent that the regulation of GTPase signaling is highly and carefully regulated. Hundreds of GTPase regulators have been identified and the G-protein signaling system is conserved well through evolution. The involvement of G-protein signaling in a wide variety of cellular activities has resulted in the study GTPases and their regulators as therapeutic targets in a number of human diseases.

Detection of Human and Mouse BIG1/ARFGEF1 by Western Blot (h&m) and Immunoprecipitation (h). Samples: Whole cell lysate from HeLa (5, 15 and 50 mcg for WB; 1 mg for IP, 20% of IP loaded), 293T (T; 50 mcg) and mouse NIH3T3 (M; 50 mcg) cells. Antibody: Affinity purified rabbit anti-BIG1/ARFGEF1 antibody (Cat. No. A300-998A) used for Western Blot at 0.1 mcg/ml (A) and at 1 mcg/ml (B) and used for Immunoprecipitation at 3 mcg/mg lysate (B). For blotting immunoprecipitated BIG1/ARFGEF1, ReliaBLOT® Reagents and Procedures (Cat. No. WB120) were used. Detection: Chemiluminescence with exposure times of 3 minutes (A) and 30 seconds (B).

Detection of Human PDZ-RhoGEF by Western Blot and Immunoprecipitation. Samples: Whole cell lysate (5, 15 and 50 mcg for WB; 1 mg for IP, 20% of IP loaded) from HeLa cells. Antibodies: Affinity purified rabbit anti-PDZ-RhoGEF antibody (Cat. No. A301-952A) used for WB at 0.04 mcg/ml (A) and 1 mcg/ml (B) and used for IP at 3 mcg/mg lysate. PDZ-RhoGEF was also immunoprecipitated by rabbit anti-PDZ-RhoGEF antibody (Cat. No. A301-951A), which recognizes an upstream epitope. For blotting immunoprecipitated PDZ-RhoGEF, ReliaBLOT® Reagents and Procedures (Cat. No. WB120) were used. Detection: Chemiluminescence with exposure times of 30 seconds (A) and 10 seconds (B).

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