Anti phospho s6 s235 236

Cell lysates were obtained from in vitro generated lymphocytes and SDS-PAGE was performed. After the transfer to a PVDF membrane (Millipore), the samples were incubated with Abs. For the assessment of AKT and mTOR activation, Bregs and Th17 cells treated with pentanoate were analyzed with following Abs: anti-phospho-AKT^Ser437 (#9271, Cell Signaling Technology, 1:1000), anti-phospho-AKT^Thr308 (#13038, Cell Signaling Technology, 1:1000), anti-total AKT (#9272, Cell Signaling Technology, 1:1000), anti-phospho-mTOR^Ser2448 (#5536, Cell Signaling Technology, 1:1000), anti-total mTOR (#2983, Cell Signaling Technology, 1:1000), anti-phosphoS6K^T389 (#9205, Cell Signaling Technology, 1:1000), anti-total S6K (#9202, Cell Signaling Technology, 1:1000), anti-phosphoS6^S235/236 (#2211, Cell Signaling Technology, 1:1000), and anti-total S6 (#2217, Cell Signaling Technology, 1:1000). The original uncropped scans of all western blots are shown in Supplementary Fig. 8.

Anti-phospho-S6 (S235/236, 1:500), anti-S6 (1:1000), anti-phospho-4E-BP (T37/46, 1:500), anti-4E-BP (1:1000), anti-phospho-STAT3 (Y705, 1:500) and anti-STAT3 (1:500) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Actin (1:10,000) antibodies, phosphatase Inhibitor Cocktails 2 and 3, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO). The nitrocellulose membrane was purchased from EMD Millipore (Billerica, MA, USA). Enhanced chemiluminescence (ECL) was purchased from GE Healthcare (Pittsburg, PA). Donkey anti-rabbit and donkey anti-mouse horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch (West Grove, PA). AMV reverse transcriptase was purchased from Promega (Madison, WI). SYBR Green PCR Master mix was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). EDTA-free complete mini Protease Inhibitor Cocktail was purchased from Roche (Indianapolis, IN). NuPAGE Bis-Tris precast gels and Phosphate buffered saline (PBS) were purchased from Life Technologies (Grand Island, NY). Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Rockford, IL). ProSignal Blotting Film was purchased from Genesee Scientific (El Cajon, CA). Ethyl alcohol (190 proof) was purchased from VWR (Radnor, PA).

Anti-phospho-Akt T450 was purchased from Abcam Technology. Anti-phospho-Akt S473, anti-pan-Akt, anti-phospho-S6 S235/236, and anti-β-actin were purchased from Cell Signaling Technology. Reagents used for flow cytometry were BD Pharmingen FITC Annexin-V Apoptosis Detection Kit I purchased from BD company. All reagents were used at a 1 : 100 dilution of the stock from the same company.