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Figure 3


Fig. 3. Analysis of K8 phosphorylation and dephosphorylation by two-dimensional gel electrophoresis. HT29 cells were cultured in isosmotic or hyposmotic conditions for 6 hours, solubilized by 1% Emp in PBS (4°C, 60 minutes), followed by immunoprecipitation of K8 and K18. Precipitates were analyzed in duplicate by isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension. One gel was then stained with Coomassie Blue and the second was transferred to a PVDF membrane for immunoblot analysis using anti-K8 Ser431-P antibodies. After blotting analysis, the PVDF membrane was stained with Coomassie Blue (not shown) in order to assign the indicated K8 charged isoforms.





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