Akt1 is required for p21 stabilization in TGCs, and Akt1 phosphorylates p21 at T140.

<p>(A) C-terminal sequence of mouse p21 protein with the putative Akt1 phosphorylation site (RKRRQTS) and the essential RKR nuclear localization signal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097434#pone.0097434-RodriguezVilarrupla1" target="_blank">[44]</a> in bold face. (B) Extracts of TSCs and TGCs were assayed by Western immuno-blotting using antibodies specific for Akt1, p21, p27 and p57 proteins. The phosphorylated form of p21 was recognized as a p21 protein that migrated slower than unphosphorylated p21, and by its reaction with an anti-p21 antibody specific for Thr-145 phosphorylation in human p21. (C) After three days of FGF4 deprivation, TGCs were stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA (gray) and with anti-Akt1 antibody (green). (D) Wild-type p21 and two p21 mutant forms with a T140V or a S141A substitution were tested as substrates for phosphorylation by Akt1 <i>in vitro</i>. (E) NIH3T3 fibroblasts were co-transfected with a plasmid expressing the tetracycline repressor and a plasmid encoding the indicated p21 protein whose expression was regulated by a tetracycline inducible promoter. Each protein carried a [His]₆-cMyc-epitope tag fused to its C-terminus. After 24 hours of transfection, the cells were cultured for 18 hours in the presence of tetracycline (1 µg/ml) in order to induce expression of the indicated recombinant p21 protein. Cells were then harvested at 0, 6, 12 and 24 hours after release, and extracts were analyzed for the indicated protein by Western immuno-blotting using a Myc-Tag specific antibody. Wild-type (wt) p21 and the T140V (T/V) and S141A (S/A) p21 mutants, as well as a double mutant (T/V+S/A) were tested. Actin served as a loading control in each case.