Figure 3

Interaction of Keratin 23 with 14-3-3ε. A) Endogenous 14-3-3ε and 14-3-3γ expression levels of Smad4 re-expressing and Smad4 negative SW480 cells. 20 μg whole cell lysates derived from Smad4 re-expressing and Smad4 negative cells were subjected to SDS-PAGE. Flag-tagged KRT23 and VSV-G-tagged 14-3-3ε were transfected into HEK 293T cells as indicated. B) Confirmation of KRT23-14-3-3ε interaction: cell lysates were immunoprecipitated with anti-Flag antibody and blotted as indicated. C) KRT23 expression leads to cytoplasmic sequestration of 14-3-3ε: following fractionation into cytoplasmic and nuclear fractions, proteins were subjected to Western blot analysis with the indicated antibodies. Anti-lamin B and anti-β-tubulin were used as marker proteins for the purity of the fractions. C, cytoplasmic fraction, N, nuclear fraction. A representative blot from three independent experiments is shown.