S6k1 inhibitor pf 4708671

Climbing assays were carried out according to previously described methods (Ng et al. 2009 (link)). In general, 30 flies per group were used for the assay and the experiment was replicated with three different sets of flies. To study the effects of drugs, flies were fed with cornmeal-agar medium supplemented with 250 μM C2 Ceramide (N-acetyl-d-sphingosine, Sigma-Aldrich), 250 μM Fingolimod (FTY720), HCl (Selleckchem) or 250 μM S6K1 Inhibitor (PF-4708671, Tocris) at day 25 post-eclosion till day 50.

Lymph node (LN) OT-I T cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, l-glutamine, antibiotics, and 50 μM 2-ME. SIINFEKL (N4), SIITFEKL (T4) and SIIGFEKL (G4) peptides (Peptide Synthesis) were added to culture media at the concentrations stated in figure legends. In some experiments, cells were cultured in the presence of 100 nM rapamycin or 10 μM S6K1 inhibitor PF-4708671 (both Tocris Bioscience). These conditions have previously been optimized for the selective inhibition of target kinases by the drugs (28 (link)–30 (link)). For CTL generation, OT-I T cells were stimulated with 10 nM N4 for 2 d, washed, and then differentiated in the presence of either recombinant human IL-2 or mouse IL-15 (both PeproTech) for an additional 4d. For cytokine recall responses, in vitro–generated CTLs or ex vivo polyclonal splenic T cells from Lm-Ova–infected mice were restimulated with peptide for 4 h in the presence of 2.5 μg/ml brefeldin A (Sigma-Aldrich).