Reagents used in the experiment

All the materials for tissue culture were purchased from Hyclone (Cramlington, NE, USA). Plasticware was obtained from Nunc (Roskilde, Denmark). EGF was purchased from Immuno Tools GmbH (Friesoythe, Germany). Gefitinib was obtained from AstraZeneca. Antibodies and Anti-PTENwere purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless otherwise indicated. PTEN antibody has been purchased from Sigma Chemical Co. (St Louis, MO, USA). Antibodies against phosphorylated forms of EGFR, HER2 and ERK1/2 were obtained from Biosource International (Camarillo, CA, USA). Akt/protein kinase B (PKB) kinase activity was performed using a non-radiactive assay kit (StressXpress AKT/PKB Elisa kit) which was purchased from Stressgene Bioreagents (Victoria, BC, Canada) on cell lysates after partial purification on a MonoQ exchange column (2 mg protein in 1 ml column) with 10 mM MOPS, pH 7.2, 25 mM -glycerophosphate, 5 mM EGTA, 2 mM EDTA, 2 mM sodium orthovanadate and 2 mM dithiothreitol and eluted using 12 ml linear NaCl gradient (0–0.8 M NaCl) with a flow rate of 0.5 ml/min. We collected fractions between 0.25 and 0.5 ml for analysis of Anti-PTEN Antibody.

 

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