Endogenous phosphorylated Akt accumulated in the nuclei of HCC cells
Expression and cellular localization of endogenous Akt1 and phosphorylated Akt1 in HCC cells. (A) Immunocytochemistry for phospho-AktS473 (a,b), total Akt (c,d), phospho-FKHR (e,f) in SMMC-7721 Sotalol hydrochloride treated with or without wortmannin (10 ng/ml). (B) Phospho-AktS473 and total Akt in Huh-7 (a,c) and Hep3B (b,d) cells illustrated by immunocytochemical analysis. (C) Western blotting analysis of phospho-AktS473, phospho-AktT308, and total Akt from nuclear (nuc) and cytoplasmic (cyto) subfraction of SMMC-7721, Huh-7 and Hep3B HCC cell lines. The purity of the fractions was verified by anti-Lamin A/C and anti-tubulin antibodies. Original magnifications: A (a, b, c, d, e, and f), B (a, b, c, and d): 400×.
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Ectopic expression of GFP tagged Akt1 and its mutants distributes both in nucleus and cytoplasm of HCC cell
The cellular localization of GFP tagged Akt1 and its mutant Akt1-AA-GFP was further studied. Following transfection with pEGFP-N1, Akt1-WT-GFP or Akt1-AA-GFP, SMMC-7721 cells were photographed by laser confocal scanning (LCS) microscope. As shown in Fig. 2A, Akt1-WT-GFP and Akt1-AA-GFP both distributed in the nucleus and cytoplasm. We further measured the fluorescence intensity in nucleus and cytoplasm of cells transfected with Akt1-WT-GFP or Akt1-AA-GFP, and the ratios of nuclear/cytoplasmic intensity were nearly equal (0.97 ± 0.11 and 0.97 ± 0.03, respectively). Both of them were little lower than that of GFP (1.02 ± 0.02) (Fig. 2B).