Cp 690550

BV-2 cells were plated 60–70% confluent and treated with 200 U/mL of IFNγ (R&D Systems) for 24–72 h. Primary microglia were plated at a density of 5 × 10⁵ cells/ml and treated with 1000 U/mL of recombinant rat IFNγ (R&D Systems). Immunoproteasome inhibition was achieved using 100 nM ONX-0914 (UBPBio) for 24 h. Jak3 inhibition was achieved using 1 μM of CP-690550 (InVivoGen) or JAk2 inhibition using AZD1480 simultaneous with IFNγ treatment. Priming experiments were performed by plating 1.0 × 10⁴ BV-2 cells or primary microglia (5 × 10⁵ cells/ml) in a clear 96-well plate. The following day, cells were treated in the following manner: 10 μl PBS, IFNγ, 100 nM ONX-0914 and IFNγ + 100 nM ONX-0914. 24 h later, media and treatments were removed and replaced with normal culture media in the presence or absence of 100 ng/mL LPS for an additional 24 h.

Apo E^−/− mice on a C57BL/6 background and their wild type littermates (4-week-old) were purchased from Peking University Laboratory Animal Center and maintained under a specific pathogen-free condition. Experiments and animal care were performed according to protocols approved by Hangzhou Normal University Institutional Animal Care and Use Committee. EL-4 T lymphoma was obtained from ATCC. INF-γ, IL-6, IL-17A and TGF-β1 ELISA kits were purchased from eBioscience. Cytofix/Cytoperm kit was obtained from BD Bioscience. CP690550 was purchased from Invivogen. [methyl-3h] thymidine was from GE Healthcare Life Sciences.