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. 2005 Jan 8;6(1):4. doi: 10.1186/1465-9921-6-4

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Copyright © 2005 Chiba et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Representative photographs showing phosphorylation of myosin light chain (MLC) in acetylcholine (ACh)-stimulated bronchi obtained from the sensitized control (Cont) and repeatedly ovalbumin-challenged (OA) mice. Isolated bronchial tissues were incubated for 10 min in the absence (non-stimulated; NS) or presence of 10^-3M ACh (see Methods). The electrophoretically separated proteins on gels were stained by Pro-Q Diamond dye, which can detect phosphorylated proteins specifically and quantitatively. After detection of phosphorylated proteins, immunoblotting for MLC was performed to detect total (phosphorylated and non-phosphorylated) MLC. The respective Pro-Q Diamond dye-positive band (bottom panel), which has same molecular weight with MLC visualized by immunoblotting (middle panel), in each sample was determined as phosphorylated MLC (p-MLC). The ACh-induced phosphorylation of MLC was augmented in the OA-challenged mice whereas the total MLC levels were equal to the control.