N-Cadherin Anti-Apoptotic Signaling separated on 4–12% SDS-PAGE, transferred onto nitrocellulose mem- branes, and immunobloted with antibodies to phospho-specific Ser473 Akt, total Akt and b-actin. The relative densities of phospho-Akt were normalized to total Akt, and the relatives densities of Akt were normalized to b-actin. Results are expressed as the mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0033206.g006 mobility shift of Bim-EL and was correlated with the expected phosphorylation of Erk1/2 (Fig. 10B). This mobility shift of Bim- EL observed in GT1-7 cells was quite similar to the one observed by Luciano et al. in different cell lines and tissues as a result of Bim-EL phosphorylation [33]. At early time points (4 hours), we detected the same mobility shift of the Bim-EL protein in cells spread on N-cad which was abrogated by U0126 treatment (Fig. 10C). These results suggest that upon N-cadherin engage- ment, Bim-EL is phosphorylated by Erk1/2 kinases. Figure 6. Akt is not involved in N-cadherin-mediated GT1-7 cell survival. Serum-starved cells were seeded at low density on PL or N- cad and grown for the indicated time in serum-free medium (A, C). In (B), confluent cell cultures were subjected to a calcium switch (see ‘‘Experimental Procedures’’). Equivalent amount of cell lysates was Figure 7. Erk1/2 phosphorylation is up-regulated by N- cadherin engagement. Serum-starved GT1-7 cells were seeded at low density on PL or N-cad and grown for the indicated time in serum- free medium (A); alternatively confluent GT1-7 cell cultures were subjected to a calcium switch (B). Equivalent amount of proteins were blotted onto nitrocellulose membranes that were incubated either with anti-phospho Erk1/2, anti-total Erk1/2, or anti-a-tubulin. The relative densities of phospho-Erk1/2 were normalized to total Erk1/2 (A) or to a- tubulin (B). Histograms present the data of three independent experiments (mean 6 SD). doi:10.1371/journal.pone.0033206.g007 PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33206