In the Absence of Arkadia, P-Smad2 Is More Stable and Correctly Localized in the Nucleus

<div><p>(A) Western blot of 8.5 dpc wild-type and <i>Akd</i>−/− embryo extracts. Each lane contains total protein extracts from three embryos. Note that P-Smad2 levels are at least 2-fold higher in mutants than in wild-type.</p> <p>(B and C) Western blot of extracts from <i>Akd−/−</i> and wild-type ES cells cultured with Activin for different intervals (in minutes (') and hours (h). Note that P-Smad2 is more stable in mutant than in wild-type cells. Loading control: PCNA, proliferation cell nuclear antigen.</p> <p>(D) Densitometry analysis of the bands on the blots in (C) showing the trend in P-Smad2 levels normalized against Smad2 where <i>Akd−/−</i>ES cells treated with Activin for 45 min is taken as 100%.</p> <p>(E) Western blot analysis showing P-Smad2 and Smad2 levels in ES cells stimulated with Activin for 1 h (1 h Act) and subsequently treated for different time points with H7 inhibitor as indicated.</p> <p>(F) Densitometry analysis of the blots in (E) showing the trend in P-Smad2 levels normalized against Smad2 where <i>Akd−/−</i> and wild-type ES cells treated with Activin for 1 h are represented as 100%.</p> <p>(G) Western blot showing P-Smad2 and Smad4 protein distribution in cytoplasmic and nuclear fractions of three different wild-type and <i>Akd−/−</i>ES cell lines as indicated. 30 μg of each protein sample was loaded for analysis. Histone H3, a nuclear protein, was used as a control for fractionation. C, cytoplasmic; N, nuclear.</p> <p>(H) Immunofluorescence with anti-P-Smad2 and anti-Smad2/3 (red) antibody on <i>Akd −/−</i> and wild-type ES cells treated with Activin A for 1.5 h prior to fixation, indicating no difference in the localization of P-Smad2 in the absence of Arkadia. Note the nuclear accumulation of P-Smad2, Smad2/3, and Smad4 in <i>Akd</i>−/− cells. White bar, 12 μm. WT, wild-type.</p>