Na3vo4

For immunoblotting, cells were lysed using RIPA buffer supplemented with 1 mM Na₃VO₄ (Santa-Cruz Biotechnology, Dallas, USA) and a protease inhibitor cocktail (P8340, Sigma-Aldrich, St.Louise, Missouri, USA). DC Protein Assay (Bio-Rad, Hercules, USA) was employed to assess protein concentration, using bovine serum albumin as the standard. Protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham™ Protran™ Premium, GE Healthcare, USA). Blocking was performed by incubating membranes for 1 h with 5% BSA in TBS-T (0.1% Tween-20) and incubated O/N at 4 °C with primary antibodies. The full list of the antibodies used is provided in Supplementary table 1. Eventually, membranes were incubated for 1 h with horseradish peroxidase-coniugated secondary antibodies (mouse or rabbit) and protein bands were detected by chemiluminescence (Amersham™ ECL™ Detection Reagents).

Starved and treated cells were lysed using RIPA buffer supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1 mM Na₃VO₄ (Santa-Cruz Biotechnology, Dallas, USA). Protein concentration was determined by DC Protein Assay (Bio-Rad, Hercules, USA), using bovine serum albumin as the standard. Protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Amersham™ Protran™ Premium, GE Healthcare, USA). Membranes were blocked for 1 h using 5% BSA (Sigma-Aldrich) in TBS-T (0.1% Tween-20) and incubated O/N (4 °C) with primary antibodies diluted in the blocking solution. Finally, membranes were incubated with horseradish peroxidase-coniugated secondary antibodies against mouse or rabbit for 1 h and protein presence was detected by chemiluminescent reaction (Amersham™ ECL™ Detection Reagents). Bands quantification was performed using Image Lab software. Details about the antibodies employed for proteins detection are reported in Supplementary Information.

CD4⁺CD45RA⁺ cells were plated at 2 × 10⁶ cells per condition for Western blotting. The cells were lysed with lysis buffer containing 2.5 mM sodium pyrophosphate, 1 mM NA3VO4, and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology). The cell lysates were resolved with 5–15% gradient SDS-PAGE (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk in TBS (20 mM Tris and 500 mM NaCl) and 0.1% Tween 20 at room temperature for 1 h followed by overnight incubation at 4°C with primary Abs against IL-1RI (Abcam), IRF4 (Santa Cruz Biotechnology), RORc (Abcam), β-actin (Sigma-Aldrich). Secondary HRP-conjugated Ab (Santa Cruz Biotechnology) was added at a dilution of 1/2000 for 1 h and the protein bands were detected with an ECL Detection System (Santa Cruz Biotechnology).