Anti pt308 akt

Cells were scraped from culture plates and incubated for 20 min in ice‐cold lysis buffer containing protease inhibitor cocktails. Nuclear and cytoplasmic fractions were prepared as described previously.^[29 (link)
^] Total protein (10 µg) was separated by SDS‐PAGE and transferred to a PVDF membrane (Bio‐Rad). The membrane was incubated with primary antibodies, and the protein was visualized with ECL (HRP) (Millipore). The following antibodies were used for western blot analysis: anti‐Pten (Cell Signaling Technology, 9188), anti‐β‐actin (Immunoway, YM3028), anti‐p‐T308‐Akt (Cell Signaling Technology, 13038), anti‐p‐S473‐Akt (Cell Signaling Technology, 4060), anti‐Akt (Cell Signaling Technology, 4691), anti‐cTnT (Abcam, ab8295), anti‐Dnmt3l (Cell Signaling Technology, 13451), anti‐Dnmt3b (Cell Signaling Technology, 48488), anti‐Dnmt3b (ab122932, Abcam), anti‐Igf1r (Cell Signaling Technology, 3027), anti‐p‐S253‐FoxO3a (Abcam, ab47285), anti‐p‐T32‐FoxO3a (Cell Signaling Technology, 9464), anti‐FoxO3a (Cell Signaling Technology, 2497), anti‐Phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4370), anti‐ Insulin Receptor β (Abcam, ab69508), anti‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4695), anti‐β‐Tubulin (Cell Signaling Technology, 2146), and anti‐Lamin A/C (Cell Signaling Technology, 4777).

The sources of antibodies and reagents are as followed: anti-pY291 Fas (prepared as previously described¹³ and validated for immunoblotting¹³ and flow cytometry Fig. S8); anti-Src, anti-Yes (R&D Systems); anti-human Fas (C20, for immunoblotting) and anti-Cyclin D1 (Santa Cruz); anti-Fas (Apo1–3, for co-immunoprecipitation) (Enzo); anti-EGFR, anti-pY1068 EGFR, anti-pY705 STAT3, anti-STAT3, anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pErk1/2, and anti-Histone H3 (Cell Signaling Technology); anti-Erk1/2 (Sigma); anti-GAPDH (Calbiochem); horseradish peroxidase conjugated anti-rabbit and anti-mouse (Jackson ImmunoResearch); anti-mouse IgG-Alexa Flour 488 (Life Technology); Recombinant human EGF (Gibco), recombinant amphiregulin (R&D Systems); protein G sepharose beads (Zymed Laboratories); WST-1 (Interchim); Matrigel (BD Bioscience). Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection according to the manufacturer’s protocol for reverse transfection.

The Akt1/2 knockout HCT116 colon cancer cell line was given as a gift from Dr Bert Vogelstein (Johns Hopkins University; Ericson et al., 2010 (link)). Cells were cultured in McCoy’s 5A (Gibco) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco) at 37°C and 5% CO₂. When the cell confluence reached around 70% in six-well plates, the medium was changed with McCoy’s 5A (Gibco) having 5% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco), and the cells were transfected with 1.5 μg of pcDNA3.1-Flag-HA-Akt plasmid or 1.5 μg (3.0 μg for Y18A mutant) of pcDNA3.1-Myr-HA-Akt plasmid complexed with 3 μL Lipofectamine 3000 (Invitrogen) and 3 μL P3000 reagent (Invitrogen) in Opti-MEM medium (Gibco) at 37°C and 5% CO₂. Seventy-two hours after transfection, the cells were washed with cold PBS and lysed by adding 150 μL RIPA buffer (Cell Signaling Technology) containing ×1 complete protease inhibitor tablet (Thermo Fisher Scientific) and ×1 PhosStop tablet (Roche), and 1 mM PMSF for 10 min at 4°C. Thirty μg of total protein (BCA assay) was loaded on an SDS-PAGE gel. Membrane transfer and western blotting were carried out as described above with 1:1000 dilution for primary antibodies: anti-Akt, anti-pT308 Akt, anti-Foxo1, anti-Foxo3a, and anti-pT24 Foxo1/pT32 Foxo3a, anti-GAPDH (Cell Signaling Technology).