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. 2007 Nov 6;2008:362741. doi: 10.1155/2008/362741

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Copyright © 2008 Yan-Ping Dai et al.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Assay of the phosphocofilin (P-CF) content in canine PA rings and cultured PASMCs by IEF. Total protein from freshly dissected PA rings (panel (a)) and undifferentiated and differentiated cells (panel (b)); recombinant CF control (rCF) were resolved by IEF electrophoresis on gels containing 1% of each ampholyte pH 6/8 and 7/9. For CF dephosphorylation, protein extracts were incubated with calf intestinal alkaline phosphatase (AP) prior to IEF electrophoresis. Protein was transferred onto nitrocellulose membranes and membranes were probed with a cofilin antibody that recognizes both unphosphorylated (CF) and phosphocofilin (P-CF). Immunoreactive bands were quantified by densitometry, and treatment-dependent changes of the P-CF/CF ratio were presented relative to untreated controls (Ctr). Vertical strips of gels (0.5 cm wide) were cut into 1 cm long segments and soaked in deionized water before assay of pH (pH bar in panel A). Panel (c): CF expression is upregulated in proliferating PASMCs (cultured in 10% NCS), compared to differentiated cells (cultured for 4 days in 0% NCS).