P gsk3α β

TNF was purchased from Sigma-Aldrich (Darmstadt, Germany) and PMSF (a protease inhibitor applied for validation experiments) from Roth (Karlsruhe, Germany). Inhibitor experiments were performed using the GSK3α/β inhibitor Kenpaullone (Sigma-Aldrich). For Western blotting, antibodies specific for CD44 (156-3C11), p-C/EBPβ (Thr235), GSK3β (27C10), IDO (D5J4E), MARCKS (D88D11), NFKB1-p105/p50, NFKB2-p100/p52, p65 (L8F6), RELB (C1E4), VIM (5G3F10), p-VIM (Ser56; all from Cell Signalling, Danvers, MA, USA), p-CD44 (Ser706; Santa Cruz Biotechnology, Santa Cruz, CA, USA), C/EBPβ (E299; Abcam, Cambridge, UK), KYNU (Biozol, Eching, Germany), p-MARCKS (Ser159), p-RELB (Ser573; Thermo Fisher), p-GSK3α/β (Tyr279/Tyr216), actin (11C), GAPDH, and TBP (Sigma-Aldrich) were used. Horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signalling or Vector Laboratories (Burlingame, CA, USA). All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza, Basel, Switzerland).

Protein samples were prepared by lysing (Cell signaling lysis buffer, #9803) and sonicating (Bandelin Sonopuls, Berlin, Germany) of the cells and quantified according to Bradford’s method68 (link). SDS-PAGE was performed with a 10% (>40 kDa) or 16% (<40 kDa) gel and 30 μg protein extract per lane. The separated proteins were transferred to a polyvinylidene fluoride membrane by semi-dry western blotting, followed by incubation with different antibodies. The following antibodies by Cell Signaling were used with a dilution of 1:1000: p-Akt (#9271), Akt (#9272), p-p70S6K (#9205), p-S6 (#2211), p-4E-BP1 (#9451), p-GSK3αβ (#9331), GSK3β (#9315), cleaved Caspase 3 (#9664), PARP (#9542), p-AMPK (#2531), AMPK (#2532), anti-rabbit (#7074) as well as α-Tubulin by Sigma-Aldrich (#T9026). All protein samples were generated for immunoblot detection in 3 independent experiments.