Nampt

T cells were sorted from splenocytes using flow cytometry and used for RNA extraction and qPCR. 3-5 animals were pooled for isolation of every sample, and this was repeated 2-3 times. RNA extractions were conducted using standard TRIzol methodology following manufacturer’s instructions (Qiagen). Extracted RNA was quantified, diluted to a total of 2 μg, and synthesized into cDNA using Superscript II (Invitrogen, Burlington, ON). SsoAdvanced Universal SYBR Green Supermix (Biorad catalog no. 1708882) was used for qPCR and run on the BioRad CFX96 mice for amplification and quantification. Gene-specific primers for murine Ifna2, Ifnr1, Ifnr2, Nampt, Kynu, Kyat1, Qprt, Nmrk1, Nmnat1, Nmnat2, Nmnat3, Gapdh and Hprt were purchased from Invitrogen (Table S2). The data from the qPCR were collected and analyzed using Livak and Schmittgen’s 2^-ΔΔCT method (65 (link)). The fold change was calculated by first normalizing the quantification cycle (Cq) of the indicated gene against Gapdh or Hprt followed by a comparison against the respective controls and bar graphs were generated using GraphPad.

The protein content of each tissue was quantified by BCA assay, and 20 μg of protein from each sample was separated on a 10–12% Bis-Tris gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The proteins were then transferred to a nitrocellulose blotting membrane (GE Healthcare, NJ, USA), blocked with 5% skim milk (BD Difco, Franklin Lakes, NJ, USA), and incubated overnight at 4 °C with the following primary antibodies: GAPDH, adiponectin, chemerin, and TNF-α (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); nampt (Invitrogen, Waltham, MA, USA); TLR4, MYD88, IL-1β, IL-6, and IF-γ (Cell Signaling Technology, Inc., Danvers, MA, USA); and NOD1 and NOD2 (Abcam, Cambridge, UK). The blots were then probed with horseradish peroxidase-labeled secondary antibodies (Solarbio Life Sciences & Technology Co., Ltd., Beijing, China) for 60 min at room temperature and visualized by chemiluminescence using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific). The intensity of the immunoreactive bands was detected using a chemiluminescence imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).