The monoclonal antibody B4/7 recognizes human and canine guanine nucleotide exchange factor H1 (GEF-H1). GEFH1 is an ~110 kDa protein belonging to the Dbl family of proto-oncogenes. GEF-H1 can activate the small GTPase RhoA, but not Rac1 or Cdc42. Rho family GTPases are central regulators of epithelial tight junctions and the cytoskeleton. GEF-H1 can associate with different cytoskeletal structures, namely microtubules and the actin cytoskeleton. It has also been proposed to mediate crosstalk between the two types of filaments.
Localization of GEF-H1 differs between cell types. In MRC-5 fibroblast cells, GEF-H1 localizes to stress fibers. In epithelial cells, GEF-H1 is associated with apical tight junctions and involved in regulating paracellular permeability of small hydrophilic tracers. Furthermore, its subcellular localization changes in mitotic cells, where endogenous GEF-H1 is concentrated at mitotic spindles. GEF-H1 is capable of binding to the F-actin binding junctional adaptor cingulin. Binding to cingulin inhibits GEF-H1 and results in the downregulation of RhoA and inhibition of G1/S phase transition. In low confluent cultured cells, the localization of GEF-H1 is predominantly cytoplasmic. With increasing density of the cells, free GEF-H1 is sequestered at the tight junctions by cingulin.
GEF-H1 is part of the signaling pathway connecting epithelial polarity with the cell cycle, and as such involved in oncogenesis.
Frozen sections, Immuno fluorescence, Immuno precipitation, Western blot
W: A non-reduced and/or reduced sample treatment and 6-15 % gradient SDS-PAGE was used. The band size is 110 kDa (Ref1-3).
F: Cells were either fixed with methanol at -20 °C or with 3% PFA followed by permeabilization with Triton X-100.
For immunohistochemistry and Western blotting, dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.