Fk506

All activators and inhibitors were of analytical grade and freshly prepared from frozen stock solution. The stock solution of LPA was 5 mM dissolved in phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA). Pertussis toxin (PTX, Sigma-Aldrich) was dissolved at a concentration of 250 µg/mL in H₂O. U0126 as well as Wortmannin (both Sigma-Aldrich) were dissolved at 10 mM in DMSO (Roth, Karlsruhe, Germany). Gö6976 (Tocris Biosciences, Bristol, UK) was dissolved at 1 mM in DMSO and ω-agatoxin-TK (Peptanova, Sandhausen, Germany) at 100 µM in H₂O. For AlF₄⁻, 30 µM AlCl₃ and 10 mM NaF were mixed. FK506 (InvivoGen, Toulouse, France) was dissolved at 20 mM in DMSO and Cyclosporin A (Alfa Aesar, Ward Hill, MA, USA) at 1 mM in ethanol.

CD4 cells were isolated from spleen and lymph nodes by negative selection as previously described (Diehl et al., 2002 (link)). For Stat3^+/+ and Stat3^−/− mice, CD4 cells were purified by cell sorting (FACS-Aria; Becton Dickinson). CD4 cells were activated with plate-bound anti-CD3 (2C11) (5 μg/ml) and soluble anti-CD28 (1 μg/ml) (BD Pharmingen, San Diego, CA) mAbs in the presence or absence of IL-6 (50 ng/mL) (Miltenyi Biotec, Auburn, CA). Pharmacological inhibitors were added to culture 42 hr after activation and supernatants were harvested 6 hr later. APCs were purified by depleting CD4 and CD8 T cells using positive selection (Miltenyi), and followed by irradiation treatment (2000 rad). APCs and OT-II CD4 cells were co-cultured at 4:1 ratio in the presence of 5 μM OVA_323-339 peptide (Barnden et al., 1998 (link)) with or without IL-6 (50 ng /mL) (Miltenyi) or anti-IL-6 (2.5 μg /mL) (BD Pharmingen).
Pharmacological inhibitors used were CGP-37157 (Tocris Bioscience, Ellisville, MO) (10 μM), CCCP (2 μM), rotenone (2 μM), antimycin (2 μM), Ru360 (10 μM), FK506 (InvivoGen, San Diego, CA) (10 nM), Stattic (10 μM).

Stock solutions of Calyculin A (1 mM, LC Labs), and FK506 (40 mM, Invivogen) were prepared in DMSO. The final concentration of DMSO in ACSF was less than 0.1%. Stock solutions of caged MNI-glutamate (80 mM, Tocris), D, L-APV (20 mM, Sigma), and TTX (2 mM, Abcom Biochemicals) were prepared in deionized water. All stocks were kept frozen at −80C0° in aliquots until use.