Akt1 specific sirnas

RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Dimethylsulfoxide (DMSO), Hoechst 33342, XAV-939, Gefitinib and YM201636 were purchased from Sigma (St. Louis, MO, USA). U0126 was purchased from Cell Signaling Technology (Beverly, MA, USA). Polyclonal anti-human β-catenin antibody, monoclonal anti-human EGFR antibody, monoclonal anti-human phospho-EGFR (Y¹⁰⁶⁸) antibody, monoclonal anti-human β-actin antibody and the corresponding horseradish peroxidase-conjugated second antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Monoclonal anti-human EEA.1, monoclonal anti-human phospho-ERK_1/2 (Thr²⁰²) antibody, polyclonal anti-human ERK_1/2 and monoclonal anti-human Lamin B antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The secondary anti-mouse or anti-rabbit antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 568 was purchased from Invitrogen (Carlsbad, CA, USA). Two different Akt1 specific siRNAs purchased from GE Dharmacon (Lafayette, CO, USA) were used: ACA AGG ACG GGC ACA TTAA (1^#siRNA), CAA GGG CAC TTT CGG CAAG (2^#siRNA).

Cells were seeded in six-well plates, and transfected with siRNA oligonucleotides using Lipofectamine^TM RNAiMAX Reagent (Invitrogen). Forty-eight hours after transfection, cells were collected. Three different AKT1 specific siRNAs (Dharmacon) were used: ACAAGGACGGGCACATTAA (siRNA #1), CAAGGGCACTTTCGGCAAG (siRNA #2), and TCACAGCCCTGAAGTACTC (siRNA #3). AKT2 siRNAs (sc-29197), AKT3 siRNA (sc-38911) and MARCKS siRNAs (sc-35857) were purchased from Santa Cruz.