Na9310

For cell lysis a cell extraction buffer was used with following ingredients: 50 mM Tris pH 7.4 (Merck), 150 mM NaCl (Merck), 2 mM ethylene glycol tetraacetic acid (EGTA; Sigma), 2 mM ethylene diamine tetraacetic acid (EDTA; Sigma), 25 mM NaF (Sigma-Aldrich), 0.1 mM Na₃VO₄ (Sigma), 0.1 mM phenylmethanesulfonylfluoride (PMSF; Sigma), 2 mg/ml leupeptin (Serva Feinbiochemika), 2 mg/ml aprotinin (Serva Feinbiochemika), 0.2% Triton X-100, and 0.3% Nonidet P-40 (Sigma). Cells were incubated for 30 min on ice. Separation of protein extracts was achived by SDS-PAGE and proteins were transferred via immunoblotting. Primary antibodies were rabbit anti-ETS2 (1:1000, GTX104527, GeneTex), rabbit anti-MYC (1:1000, sc-40; Santa Cruz) and mouse anti-β-actin (1:3000, A5541; Sigma-Aldrich). For detection anti-mouse IgG horseradish peroxidase-labeled secondary antibody (1:10000, NA9310; GE Healthcare) and ECL (Thermo Scientific/Pierce 32106) were used.

Western blot analyses were carried out as described previously [5 (link)]. Tumor tissues or cultured cells were washed with phosphate-buffered saline and homogenized in a solution with 50 mM Tris buffer, 150 mM NaCl, 1 mm ethylenediaminetetraacetic acid, 1% NP40 and proteinase (Roche Diagnostics, GmbH, Mannheim, Germany)/phosphatase inhibitors (Halt Phosphatase inhibitor cocktail, Thermo Scientific). After centrifuge at 14000 rpm for 5 minutes, lysates were used for Western blot analysis. The antibodies against MYC were used (ab32072) from Abcam, p21 (sc-6246), E2F-3 (sc-878) from Santa Cruz Biotechnology, INC (Santa Cruz, CA, USA), phosphorylated RB (S780) (#9307), RB (#9309), Bim (#2933), p-Erk1/2 (#9101) and total-ERK (#9102) from Cell Signaling, alpha tubulin (T6199) from Sigma Aldrich. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin G [21 (link)] (NA9310 GE Healthcare) or anti-rabbit IgG (NA9340 GE Healthcare) as the secondary antibody and subsequently detected by means of an ECL system (Western Lightning^® Plus-ECL, PerkinElmer, Waltham, MA, USA). Band intensities were quantified by the ImageJ software (ImageJ 1.48v; Wayne Rasband, NIH).

Western blotting was performed as described previously (Nakayama et al., 2012 (link)) on samples containing 60 wing or leg discs. Antibodies were used at the following dilutions: anti-E(z) (1:4000), anti-Pc (1:4000), anti-Pho (1:4000), anti-Pcm (gift of S. F. Newbury; 1:2000), anti-Spt16 (Dre4) (Nakayama et al., 2012; 1:4000 (link)), anti-Mbf1 (1:5000), anti-FLAG M2 (Sigma, F3165; 1:2000), anti-tubulin (Developmental Studies Hybridoma Bank, a gift of K. Saito, National Institute of Genetics, Japan; 1:5000), anti-rabbit and anti-mouse IgG-HRP (GE Healthcare, NA9340 and NA9310; 1:5000) and anti-mouse IgG-HRP.

A rat monoclonal antibody against the hemagglutinin (HA) epitope tag (11 867 423 001), a mouse monoclonal antibody against the Myc epitope tag (05–724), and rabbit polyclonal antibody against the V5 epitope tag (V8137) were purchased from Roche Applied Science (Germany), Merck Millipore (MA, USA), and SIGMA-Aldrich (MO, USA), respectively. A goat polyclonal antibody against Akt2 (AF23151) was purchased from R&D systems (MN, USA). Mouse monoclonal antibodies against Rac1 (610650) and RalA (610221) were purchased from BD Biosciences (CA, USA). A mouse monoclonal antibody against α-tubulin (T9026) was purchased from SIGMA-Aldrich. Antibodies against goat IgG, mouse IgG, rabbit IgG, and rat IgG conjugated with CF 350/543/647 were purchased from Biotium (CA, USA). A sheep polyclonal antibody against mouse IgG conjugated with horseradish peroxidase (NA9310) was purchased from GE Healthcare (UK). Insulin was purchased from Eli Lilly (IN, USA). Two siRNA duplexes against mouse Akt2, #1 (Genosys (MO, USA), Mm_AKT2_4936; 5´-GAGAUGUGGUGUACCGUG-3´) and #2 (Genosys, Mm_AKT2_4937; 5´-GACUCUUCCACAUCUGAG-3´), and a mixture of non-targeting control (NC) siRNA duplexes (Dharmacon (CO, USA), D-001206-13; Duplex 1, 5´-AUGAACGUGAAUUGCUCAAUU-3´; Duplex 2, 5´-UAAGGCUAUGAAGAGAUACUU-3´; Duplex 3, 5´-AUGUAUUGGCCUGUAUUAGUU-3´; and Duplex 4, 5´-UAGCGACUAAACACAUCAAUU-3´) were commercially obtained.