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A Novel Monoclonal Antibody Against Phosphorylated RhoA
Author(s) -
Schweinfurth Julie A.,
Dubash Adi,
Chaffin Kim,
Ellerbroek Shawn M.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb10-c
Subject(s) - rhoa , phosphorylation , blot , serine , microbiology and biotechnology , kinase , protein phosphorylation , signal transduction , chemistry , protein kinase a , biology , biochemistry , gene
Evidence indicates phosphorylation of RhoA on serine residue 188 by protein kinase A and protein kinase G inhibits and/or modifies its intracellular activity, however regulation/dynamics of RhoA phosphorylation during cellular events remains unclear. In collaboration with Pharmingen, we report here the characterization of a novel monoclonal antibody that recognizes RhoA when phosphorylated on serine residue 188. Western blotting analysis revealed an increase in exogenous RhoA phosphorylation following forskolin stimulation of NIH3T3 fibroblasts with minor binding to a non‐phosphorylatable RhoA mutant (S188A). The antibody produced a clean endogenous RhoA phosphorylation signal of ~22 kDa that varied in intensity across common cell lines when compared to total RhoA levels. The pool of endogenous phosphorylated RhoA was surprisingly static; forskolin stimulation of endogenous RhoA phosphorylation was detectable, but highly variable, while total RhoA phosphorylation was unchanged during NIH3T3 spreading on fibronectin. We conclude this monoclonal antibody has potential to be a viable tool for tracking endogenous RhoA phosphorylation by western blotting.