| Assay Method Information | |
| | MLC Phosphorylation Assay |
| Description: | Rat smooth muscle cell line A7r5 is used. The endogenous expression of ROCK results in a constitutive phosphorylation of the regulatory myosin light chain at T18/S19. A7r5 cells were plated in DMEM supplemented with 10% FCS in multiwall cell culture plates. After serum starvation overnight, cells were incubated with compounds in serum-free medium. Quantification of MLC-T18/S19 phosphorylation is assessed in 96 well-plates via ELISA using a phospho-MLC-T18/S19 specific antibody and a secondary detection antibody. Raw data were converted into percent substrate phosphorylation relative to high controls, which were set to 100%. EC50 values were determined using GraphPad Prism 5.01 software using a nonlinear regression curve fit with variable hill slope. |
| Affinity data for this assay | |
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