Figure 5

Erk1/2 signaling is required for Fgf9-mediated Cripto/Nodal activation and meiotic inhibition. (a) Representative western blot analysis of Erks and Akt phosphorylation in total and in purified Kit+ spermatogonia after short-term incubation with Kl or with Fgf9. These experiments were repeated more than 10 times with similar results. (b) Left panel: Western blot analysis of Smad2 and Erk phosphorylation and expression of Cripto and of meiotic markers (Stra8 and Scp3) in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Mek selective inhibitor U0126. Right panel: Western blot analysis of Akt phosphorylation and Stra8 expression in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Pi3k selective inhibitor LY294002. (c) Left panel: Western blot analysis of Erks phosphorylation and Stra8 expression in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Kl, in the presence or absence of the Mek selective inhibitor U0126. Right panel: Western blot analysis of Akt phosphorylation and Stra8 expression in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Kl, in the presence or absence of the Pi3k selective inhibitor LY29400. (d) Western blot analysis of Stra8 expression in cultured untreated Kit+ spermatogonia (-) and in the same cells treated overnight with all-trans Retinoic acid (AtRA) (+) in the absence or the presence of the indicated selective signaling inhibitors (U0126 for Mek; LY294002 for Pi3k; H89 for pkA; SB203580 for p38; SP600125 for Jnk). In these experiments, the Stra8 signal appears as a doublet rather than a single band, as protein extracts were separated by SDS-PAGE in 4–20% gradient gels instead than in uniform gels