ROCK Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The Rho-associated, coiled-coil containing serine/threonine kinases ROCK I and ROCK II (ROCK) are involved in the regulation of cytoskeletal activity. Activation of ROCK by binding to activated Rho-GTP results in the phosphorylation of numerous substrate proteins like regulatory myosin light chain (MLC) or the myosin binding subunit of MLC phosphatase (MYPT1). These phosphorylations lead to changes in cell morphology causing phenotypes like cellular contraction, formation of stress fibers or neurite retraction. Several of these processes play important roles in pathological conditions like cardiovascular diseases, neuronal degeneration or cancer.
ROCK1 and ROCK2
P160ROCK, KIAA0619, ROC2
The myosin light chain (MLC) in the rat aortic smooth muscle cell line A7r5 is constitutively bisphosphorylated at Thr18/Ser19 by ROCK kinases, which directly phosphorylate MLC and simultaneously prevent its dephosphorylation through inhibition of MLC phosphatase. Inhibition of ROCK kinase by specific inhibitors like Y27632 reduce the levels of phospho-MLC (see Fig.1). In the cellular assay phospho-levels of MLC-Thr18/Ser19 are quantified by direct-ELISA technique.
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
More information can be found on our website Cellular Phosphorylation Assay Services.
Reference compound IC50 for ROCK
The ROCK kinase inhibitor Y-27632 blocks ROCK and inhibits the cellular double-phosphorylation of the myosin light chain at Thr18 and Ser19 with highly reproducible IC50 values. The graph shows the result of a representative experiment.