jak1 assay kit  (BPS Bioscience)

Journal: European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences

Article Title: Exploring the Novel Role and Mechanistic Insights of Skeletal Muscle Relaxant Cyclobenzaprine Hydrochloride in Esophageal Squamous Cell Carcinoma Treatment.

doi: 10.1016/j.ejps.2025.107051

Figure Lengend Snippet: Figure 6. Flexeril inhibits ESCC progression through JAK1/STAT3 pathway suppression. (A-B) WDHD1 and STAT3 expression (protein/mRNA) in KYSE150 and Eca109 cells under STAT3 overexpression and Flexeril treatment (30 μM). (C-D) Cell viability and proliferation under combined interventions. (E-G) Migration capacity assessed by transwell/wound healing assays with rate quantification. *P < 0.05, **P < 0.01.

Article Snippet: JAK1, STAT3, phospho-JAK1, phospho-STAT3, and γ-H2AX antibodies were sourced from Cell Signaling Technology (Beverly, USA).

Techniques: Expressing, Over Expression, Migration

Journal: Burns & Trauma

Article Title: Sustained induction of IP-10 by MRP8/14 via the IFNβ–IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

doi: 10.1093/burnst/tkad006

Figure Lengend Snippet: The JAK–STAT signaling pathway is involved in the regulation of Ip-10 gene expression in MRP8/14-treated RAW264.7 cells. ( a – d ) JAK1 and TYK2 but not JAK2 or JAK3 were phosphorylated in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 5, 15, 30, 60, 120, 240 and 300 min), followed by total cellular protein extraction and immunoblot analysis of phosphorylated/total JAK1 (a), TYK2 (b), JAK2 (c) and JAK3 (d). The protein levels were quantified as the relative intensity of the protein bands on the blots. ( e ) The JAK1 inhibitor GLPG0634 blocked Ip-10 mRNA expression in RAW264.7 cells induced by MRP8/14. RAW264.7 cells were pretreated with or without GLPG0634 (500 nM) for 2 h and then stimulated with MRP8/14 (1.5 μg/ml) for 6 or 12 h. The mRNA expression levels of Ip-10 were quantified by qPCR. ( f , g ) IFNβ neutralizing Ab blocked the phosphorylation of JAK1 and TYK2 in RAW264.7 cells induced by MRP8/14. After pretreatment with the IFNβ neutralizing Ab (1 μg/ml) or isotype control IgG for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 5 h. Phosphorylated and total JAK1 (f) and TYK2 (g) were examined by immunoblotting with specific antibodies. The protein levels were quantified as the relative intensity of the protein bands on the blots. ( h ) Dynamic profile of STAT1 phosphorylation in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were stimulated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 5, 15, 30, 60, 120, 240 and 300 min), followed by total cellular protein extraction and immunoblot analysis of phosphorylated and total STAT1. The protein levels were quantified as described above. ( i ) Time-dependent STAT1 nuclear translocation in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were stimulated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 60, 120, 240, 300 min), followed by total nuclear protein extraction and immunoblot analysis of total STAT1 and lamin as a control. The protein levels were quantified as described above. ( j ) The IFNβ neutralizing Ab blocked the phosphorylation of STAT1 in RAW264.7 cells treated with MRP8/14. After pretreatment with the IFNβ neutralizing Ab (1 μg/ml) or isotype control IgG for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 5 h. Phosphorylated and total STAT1 were examined by immunoblotting with specific Abs. The protein levels were quantified as the relative intensity of the protein bands on the blots. ( k ) The STAT1 inhibitor fludarabine blocked Ip-10 mRNA expression in RAW264.7 cells induced by MRP8/14. RAW264.7 cells were pretreated with or without fludarabine (10 μM) for 2 h and then stimulated with MRP8/14 (1.5 μg/ml) for 6 or 12 h. The mRNA expression levels of Ip-10 were quantified by qPCR. The data are expressed as the mean ± SD and represent three independent experiments ( n = 3). * p < 0.05, * * p < 0.01. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, JAK1 Janus kinase 1, p-JAK1 phosphorylated Janus kinase 1, JAK2 Janus kinase 2, p-JAK2 phosphorylated Janus kinase 2, JAK3 Janus kinase 3, p-JAK3 phosphorylated Janus kinase 3, TYK2 tyrosine kinase 2, p-TYK2 phosphorylated tyrosine kinase 2, GLPG0634 JAK1 inhibitor, IFNβ Ab interferonβ neutralizing antibody, STAT1 signal transducer and activator of transcription 1, p-STAT1 phosphorylated signal transducer and activator of transcription 1, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA

Article Snippet: All specific Abs against phospho-JAK1, JAK2, JAK3, TYK2 and STAT1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Gene Expression, Protein Extraction, Western Blot, Expressing, Phospho-proteomics, Control, Translocation Assay, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Sustained induction of IP-10 by MRP8/14 via the IFNβ–IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

doi: 10.1093/burnst/tkad006

Figure Lengend Snippet: IRF7 functions downstream of the JAK–STAT pathway to mediate MRP8/14-induced Ip-10 gene expression in macrophages. ( a ) Profile of IRF7 protein expression in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 2, 4, 6, 8, 10, 12 and 14 h), followed by total cellular protein extraction and immunoblot analysis of IRF7. The protein levels were quantified as described above. ( b ) Inhibition of the JAK–STAT pathway reduced IRF7 production in RAW264.7 cells induced by MRP8/14. After pretreatment with GLPG0634 (500 nM) or fludarabine (10 μM) for 2 h, RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for 12 h. IRF7 protein expression was quantified as described above. ( c ) Nuclear translocation of IRF7 in MRP8/14-treated RAW264.7 cells. RAW264.7 cells were treated with MRP8/14 (1.5 μg/ml) for the indicated times (0, 8, 10, 12and 14 h), followed by nuclear protein extraction and immunoblot analysis of IRF7. Lamin was used as a control and the protein levels were quantified as described above. ( d ) Irf7 gene deficiency reduced late-stage Ip-10 mRNA expression in BMDMs induced by MRP8/14. BMDMs were isolated from WT or Irf7 −/− mice and treated with or without MRP8/14 (1.5 μg/ml) for 6 or 12 h. Total RNA was extracted from BMDMs by the TRIzol method and Ip-10 mRNA expression was quantified by qPCR. ( e ) Irf7 gene overexpression rescued the transcriptional activity of Ip-10 that was downregulated by STAT1 inhibitor in MRP8/14-treated RAW264.7 cells. After transfection with the Ip-10 reporter (PGL3- Ip10p ) with or without the irf7 -expressing plasmid (pcDNA3- Irf 7), RAW264.7 cells were cultured for 24 h. Then, the cells were incubated with fludarabine (10 μM) for 2 h and treated with MRP8/14 (1.5 μg/ml) for 12 h. Cell lysates were extracted for the dual-luciferase assay, and relative luciferase activities were calculated by determining the ratio of firefly and Renilla luciferase activities. The data are expressed as mean ± SD and represent three independent experiments ( n = 3). * p < 0.05, * * p < 0.01. IRF7 interferon regulatory factor-7, MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, JAK Janus kinase, STAT signal transducer and activator of transcription, GLPG0634 JAK1 inhibitor, WT wild-type, Irf7 −/− Irf7 gene deficiency, BMDMs bone marrow-derived macrophages, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA

Article Snippet: All specific Abs against phospho-JAK1, JAK2, JAK3, TYK2 and STAT1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Gene Expression, Expressing, Protein Extraction, Western Blot, Inhibition, Translocation Assay, Control, Isolation, Over Expression, Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Incubation, Luciferase, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Sustained induction of IP-10 by MRP8/14 via the IFNβ–IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

doi: 10.1093/burnst/tkad006

Figure Lengend Snippet: Chemotaxis of CXCR3 + lymphocytes was induced by activation of the IFNβ-JAK1/TYK2-STAT1-IRF7 pathway in vivo. ( a , b ) Mrp8 gene deficiency reduces the expression of Ip-10 and Ifnb mRNAs in endotoxic mice. WT mice were treated with LPS (20 mg/kg, i.p.), MRP8/14 (4 mg/kg, i.v.) or an equal volume of NS. Mrp8 ΔMC mice were treated with LPS, LPS plus MRP8/14, or an equal volume of NS. After treatment with LPS or MRP8/14 for 12 h, AMs were isolated, and Ip-10 (a) and Ifnb (b) mRNA expression was quantified by qPCR. ( c – e ) Effect of Mrp8 gene deficiency on the phosphorylation of JAK1, TYK2 and STAT1 in AMs induced by LPS. After treatment with LPS or MRP8/14 for 12 h, AMs were isolated from mice, phosphorylated and total JAK1 (c), TYK2 (d) and STAT1 (e) levels were quantified by immunoblotting. The protein levels were quantified by determining the relative intensity of the protein bands on the blots. ( f ) Effect of Mrp8 gene deficiency on IRF7 expression in AMs treated with LPS. AMs were isolated from mice treated with LPS for 12 h and immunoblotting was performed to quantify IRF7 protein expression. ( g – i ) The IFNβ-JAK1/TYK2-STAT1-IRF7 signaling pathway was involved in the transmigration of CXCR3 + lymphocytes induced by MRP8/14 in vivo . After pretreatment with or without the IFNβ neutralizing Ab (1 μg/ml), GLPG0634 (500 nM) or fludarabine (10 μM) for 2 h, BMDMs from WT or Irf7 −/− mice were treated with MRP8/14 (1.5 μg/ml) for 36 h, and cell culture supernatants were collected for the air pouch assay. After preparation of the air pouches in vivo for 5 days, the mice were i.p. administered LPS for 8 h. Then, the cell culture supernatants were injected into the air pouches and the lavage fluids were collected from the air pouches for further analysis. Wright–Giemsa staining was performed and the total number of migrated lymphocytes was counted (g). Flow cytometry was performed to analyse the proportion of CXCR3 + lymphocytes among all the migrated cells (h, i). The data are expressed as mean ± SD and represent three independent experiments ( n = 3). * p < 0.05, * * p < 0.01. CXCR3 chemokine (C-X-C motif) receptor 3 , MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, WT wild-type, Irf7 −/− Irf7 gene deficiency, Mrp8 ΔMC Mrp8 gene-deficient, LPS lipopolysaccharide, NS normal saline, IFNβ interferonβ, JAK1 Janus kinase 1, TYK2 tyrosine kinase 2, STAT1 signal transducer and activator of transcription 1, IRF7 interferon regulatory factor-7, GLPG0634 JAK1 inhibitor, IFNβ Ab interferonβ neutralizing antibody, AMs alveolar macrophages , BMDMs bone marrow-derived macrophages, qPCR quantitative real-time polymerase chain reaction, mRNA messenger RNA

Article Snippet: All specific Abs against phospho-JAK1, JAK2, JAK3, TYK2 and STAT1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Chemotaxis Assay, Activation Assay, In Vivo, Expressing, Isolation, Phospho-proteomics, Western Blot, Transmigration Assay, Cell Culture, Injection, Staining, Flow Cytometry, Saline, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Sustained induction of IP-10 by MRP8/14 via the IFNβ–IRF7 axis in macrophages exaggerates lung injury in endotoxemic mice

doi: 10.1093/burnst/tkad006

Figure Lengend Snippet: Signaling model for late-phase IP-10 induction by MRP8/14 in endotoxic mice. At the beginning of endotoxemia, MRP8/14 is released from activated neutrophils into the blood. Extracellular MRP8/14 binds to TLR4 on the surface of macrophages to induce IFNβ production and release. Then, IFNβ activates the JAK1/TYK2-STAT1 signaling pathway by binding with IFNAR, resulting in irf7 gene transcription and IRF7 protein synthesis. As a transcription factor, IRF7 promotes Ip-10 gene transcription, resulting in sustained production of IP-10 in the late phase of endotoxemia. Finally, the accumulated IP-10 attracts a large number of CXCR3 + lymphocytes to the site of infection, leading to an over-reactive inflammatory response and lung injury. MRP8/14 myeloid-related protein 8/14, IP-10 IFNγ inducible protein 10, IFNβ interferon β, JAK1 Janus kinase 1, TYK2 tyrosine kinase 2, STAT1 signal transducer and activator of transcription 1, IRF7 interferon regulatory factor-7, CXCR3 chemokine (C-X-C motif) receptor 3, IFNAR type I interferon receptor

Article Snippet: All specific Abs against phospho-JAK1, JAK2, JAK3, TYK2 and STAT1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay, Infection