Anti phospho foxo3

Whole cell lysates were generated from purified TA-DC using lauryl-maltoside (Sigma Aldrich) immunoprecipitation buffer supplemented with one protease inhibitor tablet (Roche). Lysates were either used for a direct western blot or immunoprecipitated for FOXO3 or NF-κB proteins as indicated. For immunoprecipitation, Abs were incubated with Protein G sepharose beads (Ambion) for 1 hr prior to adding cell lysates. Lysates were incubated with Ab coated beads for 4 hr at 4°C with rotation. Precipitated proteins were tested by western for co-precipitation. To confirm cellular localization of protein, cytoplasmic or nuclear fractionation was performed on lysates with the Paris protein fractionation kit (Ambion) per manufacturer's instructions. Proteins were run by electrophoresis on a 12% polyacrylamide gel and transferred to a nitrocellulose membrane using the Rapid Transfer semi-dry transfer box (BioRad). Membranes were blotted with anti-FOXO3 (R&D Systems, Cell Signaling), anti-phospho-FOXO3 (Cell Signaling), anti-NF-κB RelA, RelB, or c-rel (Cell Signaling), β-ACTIN (Sigma Aldrich), LAMIN-B1 (Abcam), anti-IκBα and anti-IKKα (Cell Signaling).

Anti-phospho-S473-Akt, anti-phospho-T308-Akt, anti-Akt1, anti-total Akt, anti-phospho-4E-BP1, anti-4E-BP1, anti-phospho-S6K1, anti-S6K1, anti-phospho-S6, anti-S6, anti-phospho-FoxO3, anti-FoxO3, anti-phospho-GSK3 α/β, anti-GSK3 α/β, anti-phospho-IGF1R, anti-IGF1R, anti-phospho-IRS, anti-IRS, anti-phospho-ERK1/2, anti-ERK1/2, anti-TSC2, anti-mTOR, anti-Rictor, and anti-phospho-tyrosine antibodies were purchased from Cell Signaling Technology. Anti-phospho-S422-SGK and anti-Grb10 antibodies were obtained from Santa Cruz Biotechnology. Rapamycin, Torin1, Ku-0063794, BKM-120, GDC-0941, AZD8931, Erlotinib, BMS754807, OSI906, PF431396, PF573228, and SB273005 were purchased from Selleckchem. Cpd22 and PDGFR inhibitor III were purchased from Calbiochem. Anti-integrin α2, anti-integrin αV, and anti-integrin α5 antibodies for flow cytometry were obtained from BD Biosciences. BTT3033 and anti-integrin α2 antibodies for immunoblot analysis were obtained from Tocris and SantaCruz Biotechnology, respectively. Anti-actin and anti-vinculin antibodies, N-methyl-2-pyrrolidone, PEG400, and lentiviral shRNA plasmids were purchased from Sigma.