Pe mhc 2

GL was purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Gentamycin (GM) was obtained from Amersco (Amersco, USA). LPS (Escherichia coli 0111:B4) and FITC-dextran (40,000 Da) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant mouse GM-CSF and IL-4 were obtained from PeproTech Inc. (RockyHill, NJ, USA). Anti-mouse antibodies FITC-CD11c, -CD40, -CD80, -CD83 and -CD86, Alexa Fluor®647-TLR2 and PE-MHC-II as well as anti-NF-κB p65 were obtained from Biolegend (San Diego, CA, USA). The ELISA kits for IFN-γ, TNF-α, NO, IL-6, IL-10 and IL-12p70 were obtained from eBioscience (San Diego, CA, USA). Antibodies against β-actin, phospho-JNK, JNK, phospho-p38, p38, IκBα, LaminB1 and HRP-conjugated anti-mouse and anti-goat IgG were purchased from SantaCruz Biotech (Santa Cruz, CA, USA), and phospho-ERK1/2 and anti-ERK1/2 antibodies were obtained from BD Pharmingen (San Jose, CA, USA). The inhibitors BAY 11-7082, SP600125, SB203580 and U0126 were purchased from Beyotime Biotechnology (Haimen, Jiangsu, China).

Single-cell suspensions were prepared from the spleen and VAT and stained with PE-MHC-II (107613, Biolegend), APC-cy7-CD11c (117323, Biolegend), FITC-CD64 (139315, Biolegend), Percp-cy5.5-CD45.2 (109827, Biolegend), APC-CD45 (147707, Biolegend), FITC-CD4 (100405, Biolegend), APC-CD8 (126613, Biolegend), PE-cy7-CD62L (104417, Biolegend), Percp-cy5.5-CD44 (103031, Biolegend), APC-cy7-FVD, or Pacific blue-DAPI. Intracellular staining was performed using PE-Foxp3 (12-5773-82, eBioscience) and antibodies against cytokines, including BV421-CD8, PE-cy7-IFN-γ (505825, Biolegend), Percp-cy5.5-IL-17A (506919, Biolegend), and APC-IL-4 (504105, Biolegend). Cells from the spleen and VAT were treated with phorbol myristate acetate (PMA, 50 ng/mL) and ionomycin (500 ng/mL) (Sigma-Aldrich) for 4.5 h. Then the cytokines in specific cell populations were analyzed. The cells were stained at 4 °C for 30 min or 1 h for surface and intracellular staining, respectively, followed by detection using the FACS Canto II (BD Biosciences) system and analysis of the data using FlowJo (Tree Star, version 10.0).