Age-matched female C57BL/6 mice (= 13) and MRL/mice (= 13) were also purchased from the Model Animal Research Center of Nanjing University and euthanized at 22C24 weeks old. IFN–induced phosphorylation of signal transducer and activator of transcription MRTX1257 2 (STAT-2) and expression of IFN-stimulated genes (ISGs) in a Lyn-PI3K-BTK-dependent manner and = 10) and female healthy donors (= 10) were obtained from Jiangsu Province Hospital of Traditional Chinese Medicine (Jiangsu Province, China). PBMCs were separated from the plasma using Ficoll centrifugation (Lymphoprep, Nycomed, Oslo, Norway) according to the standard procedures. B cells were isolated using human CD19+ B-Cell Isolation Beads, as previously described.8 The purity of B cells was consistently above 95%. For experiments, isolated human CD19+ MRTX1257 B cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and stimulated with the TLR7 ligand R848 (1 g mL?1, Enzo Life Sciences, Farmingdale, NY, USA), the TLR9 ligand CpG-2006S (0.3 M, Invitrogen, Carlsbad, CA, USA), or human IFN- (1000 U mL?1, eBioscience, San Diego, CA, USA). Mice Six- to eight-week-old female C57BL/6 (B6) mice were used to isolate splenic B cells and were purchased from the Model Animal Research Center MRTX1257 of Nanjing University. Age-matched female C57BL/6 mice (= 13) and MRL/mice (= 13) were also purchased from the Model Animal Research Center of Nanjing University and euthanized at 22C24 weeks old. The mice were maintained under specific pathogen-free conditions. All manipulations were in accordance with the institutional guidelines for animal care and used based on the Guide for the Animal Care Committee at Nanjing University. Purification of murine splenic B cells and experiment Splenic lymphocytes were isolated by Ficoll density centrifugation according to standard procedures. B cells were purified from the spleen by positively selecting B220+ B cells using B220 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and the purity of B cells was consistently above 95%. The purified B cells were cultured in RPMI 1640 medium containing 10% FBS and stimulated with the TLR7 ligand R848 (1 g mL?1, Enzo Life Sciences, Farmingdale, NY, USA), the TLR9 ligand CpG-1826 (0.3 M, Invitrogen, Carlsbad, CA, USA), mouse IFN- (1000 U mL?1, eBioscience, San Diego, CA, USA), or anti-CD180 antibody (0.2 g mL?1, eBioscience, San Diego, CA, USA). For inhibitor studies, the B cells were pretreated with dasatinib (5 M, Selleck, Houston, TX, USA), ibturinib (1 M, Selleck, Houston, TX, USA), enzastaurin (1 M, Selleck, Houston, TX, USA), LY294002 (5 M, Beyotime, Haimen, China), U0126 (3 M Beyotime, Haimen, China), SP600126 (2 M Beyotime, Haimen, China), or SB20358 (1 M Beyotime, Haimen, China) 1 h prior to stimulation with anti-CD180 or Nr4a3 IFN-. Anti-CD180 antibody and IFN- treatment Female C57BL/6 mice (= 24), 8C10 weeks old, were purchased from Model Animal Research Center of Nanjing University and were randomly divided into four groups: (a) PBS; (b) anti-CD180; (c) IFN-; (d) anti-CD180 + IFN-. The mice were injected with 100 g anti-CD180 antibody (contained in 200 l PBS) or 10 000 U IFN- (contained in 200 l PBS). All of the injections were conducted intraperitoneally (IP). Six hours later, the mice were euthanized. The spleens, bone marrow, and PBMCs were isolated and used to analyze the expression of IFIT1, MX1, and TLR7. The B220+ B cells were purified from spleen as described above. RNA isolation and quantitative real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. qPCR assays for mRNA were carried out on a StepOne Plus real-time polymerase chain reaction system or an ABI Vii 7 detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix. The 2 2?Ct method was used for real-time PCR gene MRTX1257 expression analysis. All quantification data are presented as a ratio to the GAPDH level. Western blot analysis Proteins were extracted using lysis buffer containing 10 mM Tris-HCl (pH 7.3), 150 mM NaCl, 2 mM Na3VO4, 0.4 mM EDTA, 10 mM NaF, 1 mM PMSF, 5 g mL?1 aprotinin and leupeptin, and 1% NP-40. The total lysates were resolved by 10% sodium dodecyl sulfateCpolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA, USA). After blocking, the membranes were incubated with primary antibodies against GAPDH, phos-JAK1 at MRTX1257 tyrosine 1022, total-JAK1, phos-signal transducer and activator of transcription 1 (STAT-1) at tyrosine 701, phos-STAT-1 at serine 727, total STAT-1, phos-STAT-2 at tyrosine 690, total STAT-2 and TLR7 (Cell Signaling Technology, Danvers, MA, USA, all dilutions at 1:1000) overnight at 4 C. After washing, the membranes were incubated at room temperature with the secondary antibody AffiniPure Goat Anti-Rabbit IgG (H+L) (Beyotime, Haimen, China, dilution at 1:3000). ECL Plus western blotting detection reagents (Millipore Corp) were used to visualize.