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Immunoblot analysis

AS Ana Silva
AA Afonso R. M. Almeida
AC Ana Cachucho
JN João L. Neto
SD Sofie Demeyer
MM Mafalda de Matos
TH Thea Hogan
YL Yunlei Li
JM Jules Meijerink
JC Jan Cools
AG Ana Rita Grosso
BS Benedict Seddon
JB João T. Barata
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Cells were lysed as described elsewhere.21 Equal amounts of protein (50 μg/sample) were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with the antibodies at 1:1000 dilution: p27Kip1, actin (Santa Cruz Biotechnology, Santa Cruz, CA), PTEN, and p-Akt (S473; Cell Signaling Technology, Danvers, MA). Immunodetection was performed by incubation with horseradish peroxidase–conjugated anti-mouse (1:5c000), anti-rabbit IgG (1:10c000), or anti-goat (1:5c000) (Promega, Madison, WI) and developed by enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ).

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