Foxo3 75d8

Protein extracts were prepared by lysis in RIPA buffer (300 mM NaCl, 2% IGEPAL CA-630, 1% deoxycholic acid, 0.2% SDS, 100 mM Tris-HCl pH 8.0) containing complete ULTRA protease inhibitors (Roche, Basel, Switzerland). Western blots were carried out using 30 μg total protein per sample run on NuPAGE Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes with an iBlot (Life Technologies) according to the manufacturer's protocols. Blots were probed with the following antibodies: p53 (FL-393, Santa Cruz Biotechnology, Santa Cruz, CA, USA); p19ARF (p19ARF exon 2, Rockland, Gilbertsville, PA, USA); Mdm2 (C-18, Santa Cruz Biotechnology); FoxO3 (75D8, Cell Signaling Technology); FoxO1 (C29H4, Cell Signaling Technology); and β-actin (clone AC-74, Sigma-Aldrich).

U2OS cells, were grown in DMEM supplemented with 10% foetal bovine serum. U2OS cells stably expressing FOXK2-HF (U2OS-FOXK2-HF), a control ‘empty vector’ cell line (U2OS-HF) were made and propagated as described previously (10). To create U2OS cell lines stably expressing 3xFLAG-FOXO3 (U2OS-3xFLAG-FOXO3), 3xFLAG-FOXJ3 (U2OS-3xFLAG-FOXJ3), or 3xFLAG-FOXK2(1–430)-Sso7dE35L (U2OS-3xFLAG-FOXK2(1–430)-Sso7d(E35L)) the parental line U2OS-TREX (gift from C Millar), was transfected with pOG44 (Invitrogen) together with either pAS3012 (3xFLAG-FOXO3), pAS3088 (3xFLAG-FOXJ3) or pAS4314 (3xFLAG-FOXK2(1–430)-Sso7dE35L), respectively. Hygromycin-resistant colonies were pooled and expanded.
siRNA against FOXK2 and a matched Non-targeting (NT) control, were obtained from Dharmacon and RNA interference (RNAi) was performed as described previously (15 (link)).
For visualization of FOXO3 subcellular localization, fluorescence microscopy was performed mainly as described previously (10 (link)) using these following antibodies: FLAG M2 (Sigma Aldrich, F3165), FOXO3 (75D8) (Cell Signalling), Alexa Fluor 594 donkey anti-mouse (Invitrogen) and Alexa Fluor 488 donkey anti-rabbit (Invitrogen).