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Figure 4


Fig. 4. Effect of hyposmosis on K19 and K20 phosphorylation. (A) Total cell extracts were prepared from HT29 cells (Total) or high salt extraction (HSE) was used to generate a highly enriched keratin fraction followed by analysis with SDS-PAGE and Coomassie Blue staining. (B) HT29 cells were incubated in isotonic or hypotonic medium for 6 hours, harvested, then subjected to SDS-PAGE followed by immunoblotting using antibodies to K19 or K20, or phospho-K19 or phosho-K20 (pK19 and pK20). Lanes 3 included homogenates isolated from OA-treated HT29 cells, as a positive control for the phospho-antibody reactivity. (C) Total cell lysates from iso- or hypo-treated HT29 cells (as in panel B) were analyzed by isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in second dimension then immunoblotting using anti-K19 or anti-K20 antibodies. Spot 1 is the non-phosphorylated isoform; others are phosphorylated isoforms for K19 and K20.





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