Journal: Virology

Article Title: Inhibition of porcine epidemic diarrhea virus (PEDV) replication by A77 1726 through targeting JAK and Src tyrosine kinases

doi: 10.1016/j.virol.2020.06.009

Figure Lengend Snippet: Inhibition of S6K1 and PTK by A77 1726. IPEC-DQ ( A, C, E ) and Vero cells ( B, D, F ) were left uninfected ( A, B, E, F ) or infected with 0.5 MOI PEDV ( C & D ). The cells were then incubated in the absence or presence of the indicated concentrations of A77 1726 for 12 h ( A, C, E ) or 24 h ( B, D, F ). The cell lysates were analyzed for the levels of JAK1, JAK2, STAT3, and Src phosphorylation by Western blot ( A-D ) or for total protein tyrosine phosphorylation ( E & F ).

Article Snippet: An anti-phospho-JAK1 antibody (#10-P1628-1) was purchased from American Research Products, Inc. (Waltham, MA).

Techniques: Inhibition, Infection, Incubation, Phospho-proteomics, Western Blot

Journal: Virology

Article Title: Inhibition of porcine epidemic diarrhea virus (PEDV) replication by A77 1726 through targeting JAK and Src tyrosine kinases

doi: 10.1016/j.virol.2020.06.009

Figure Lengend Snippet: STAT3 overexpression enhances PEDV replication. ( A ) IPEC-DQ cells were pre-incubated in the absence or presence of the indicated concentrations of A77 1726 for 2 h and then infected with the HLJBY strain of PEDV by incubating the cells at 4 °C for 2 h. After rinse twice, the cells were incubated in serum-free medium and incubated at 37 °C for 30 min. Cell lysates were prepared and analyzed for JAK1, JAK2, and STAT3 tyrosine phosphorylation and the level of the N protein of PEDV by Western blot. ( B ) STAT3 overexpression enhances viral protein synthesis. IPEC-DQ cells were transfected with the pcDNA3.1 empty vector or the expression vector encoding STAT3. After incubation for 24 h, the cells were infected with the indicated MOI of PEDV and then incubated for 24 h. The cell lysates were prepared and analyzed for the expression of indicated proteins by Western blot. The density of the phosphorylated JAK1, JAK2, and STAT3 bands was analyzed by using NIH Image-J software and normalized by the arbitrary units of β-actin or STAT3. * p < 0.05, ** p < 0.01, compared to the corresponding samples in the cells transfected with pcDNA3.1 with a Student's t -test.

Article Snippet: An anti-phospho-JAK1 antibody (#10-P1628-1) was purchased from American Research Products, Inc. (Waltham, MA).

Techniques: Over Expression, Incubation, Infection, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Expressing, Software

Journal: Pharmacology Research & Perspectives

Article Title: 2‐Deoxy‐ d ‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation

doi: 10.1002/prp2.940

Figure Lengend Snippet: 2‐Deoxy‐D‐glucose (2‐DG) inhibits interleukin (IL)‐6 signalling and N ‐linked glycosylation of its receptor gp130. (A) Wildtype mouse embryonic fibroblasts (MEFs) were incubated in complete medium, glucose‐depleted medium (Glc‐), or medium with 25 mM 2‐DG for 5 h and then stimulated with IL‐6 and/or soluble IL‐6 receptor (sIL‐6R) (0.6 µg/mL each) for the indicated periods. Activating phosphorylation of Janus kinases (JAK1, JAK2, and TYK2) and expression of gp130 were assessed by immunoblotting. (B) Wildtype MEFs were incubated in medium with 25 mM 2‐DG or glucose‐depleted medium for the indicated periods. Band shifts of gp130 were assessed by immunoblotting. Glycopeptidase F (GP‐F) extract was used as a control for inhibition of N ‐linked glycosylation. (C) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and whole cell extracts were precipitated with concanavalin A (ConA) agarose. ConA‐bound proteins were subjected to immunoblotting with an anti‐gp130 antibody. WCE, control whole cell extracts subjected to immunoblot analysis. (D) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and then whole cell extracts were immunoprecipitated with the anti‐gp130 antibody and subjected to ConA staining. (E) Wildtype MEFs were incubated with or without 25 mM 2‐DG for 5 h and whole cell extracts were subjected to ConA and wheat germ agglutinin (WGA) staining. (F) Wildtype MEFs were incubated in medium with 25 mM 2‐DG or 100 µg/mL cycloheximide for the indicated periods. Expression of gp130 was assessed by immunoblotting. (G) Binding of IL‐6 to its receptor in the presence or absence of 2‐DG was assessed using FITC‐conjugated IL‐6 by flow cytometry analysis of human acute monocytic leukemia cells (THP1, left panel). The same cells were subjected to immunoblotting using the anti‐gp130 antibody as described in (B)

Article Snippet: Antibodies against gp130 (1:1000; Merck Life Science), phospho‐JAK1 (1:1000, Tyr 1022/1023/p‐JAK1; Cell Signaling Technology, Danvers, MA, USA), JAK1 (1:1000; Merck Life Science), phospho‐JAK2 (1:1000; Tyr 1007/1008/p‐JAK2; Cell Signaling Technology), JAK2 (1:1000; Merck Life Science), phospho‐TYK2 (1:1000; Tyr1054/1055/pTyk2; Cell Signaling Technology), TYK2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), phospho‐STAT3 (1:1000; Tyr 705/p‐STAT3; Cell Signaling Technology), STAT3 (1:1000; Cell Signaling Technology), phospho‐STAT1 (1:1000; Tyr 701/p‐STAT1; Cell Signaling Technology), STAT1 (1:1000; Santa Cruz Biotechnology), TNFR1 (1:1000; Cell Signaling Technology), IFNGR‐α (1:1000; Santa Cruz Biotechnology), α‐tubulin (1:1000; Sigma‐Aldrich, St. Louis, MO, USA), and β‐actin (1:1000; Sigma‐Aldrich) were used for immunoblot analyses.

Techniques: Incubation, Expressing, Western Blot, Inhibition, Immunoprecipitation, Staining, Binding Assay, Flow Cytometry

Journal: Pharmacology Research & Perspectives

Article Title: 2‐Deoxy‐ d ‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation

doi: 10.1002/prp2.940

Figure Lengend Snippet: 2‐Deoxy‐D‐glucose (2‐DG) inhibits the functions of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and interferon (IFN)‐γ. (A) HeLa cells were transfected with Stat3 Luc or NF‐κB Luc reporter plasmids or phRL‐TK (internal control). After 24 h, cells were treated with or without 25 mM 2‐DG for 8 h and then with 0.4 µg/mL IL‐6 with soluble IL‐6 receptor (sIL‐6R) (Stat3 Luc), 50 ng/mL IL‐1β (NF‐κB Luc), or 100 ng/mL TNF‐α (NF‐κB Luc) for 4 h. After stimulation, relative luciferase activity was evaluated. Results were analysed using one‐way ANOVA followed by Tukey's post hoc test. **** p < .0001, *** p < .001, ** p < .01 and * p < .05. n.s.; not significant. Graphs are presented as the mean ±s.d. (n = 3). (B) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. The band shift of TNF receptor 1 (TNFR1) was determined by immunoblotting. Glycopeptidase F (GP‐F) extract was used as a control for inhibition of N ‐linked glycosylation. (C) Binding of TNF‐α to its receptor in the presence or absence of 2‐DG was determined using FITC‐conjugated TNF‐α by flow cytometric analysis of THP1 cells (left panel). The same cells were subjected to immunoblotting using an anti‐TNFR1 antibody as described in (B). (D) Wildtype mouse embryonic fibroblasts (MEFs) were stimulated with 3000 U/mL IFN‐γ for the indicated times. Activating phosphorylation of Janus kinases (JAK1, JAK2, and TYK2) was assessed by immunoblotting. (E) HeLa cells were incubated in medium with 25 mM 2‐DG for the indicated periods. Band shift of IFN‐γ receptor α‐chain (IFNGRα) was assessed by immunoblotting. Tunicamycin and GP‐F extracts were used as controls for inhibition of N ‐linked glycosylation

Article Snippet: Antibodies against gp130 (1:1000; Merck Life Science), phospho‐JAK1 (1:1000, Tyr 1022/1023/p‐JAK1; Cell Signaling Technology, Danvers, MA, USA), JAK1 (1:1000; Merck Life Science), phospho‐JAK2 (1:1000; Tyr 1007/1008/p‐JAK2; Cell Signaling Technology), JAK2 (1:1000; Merck Life Science), phospho‐TYK2 (1:1000; Tyr1054/1055/pTyk2; Cell Signaling Technology), TYK2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), phospho‐STAT3 (1:1000; Tyr 705/p‐STAT3; Cell Signaling Technology), STAT3 (1:1000; Cell Signaling Technology), phospho‐STAT1 (1:1000; Tyr 701/p‐STAT1; Cell Signaling Technology), STAT1 (1:1000; Santa Cruz Biotechnology), TNFR1 (1:1000; Cell Signaling Technology), IFNGR‐α (1:1000; Santa Cruz Biotechnology), α‐tubulin (1:1000; Sigma‐Aldrich, St. Louis, MO, USA), and β‐actin (1:1000; Sigma‐Aldrich) were used for immunoblot analyses.

Techniques: Transfection, Luciferase, Activity Assay, Incubation, Electrophoretic Mobility Shift Assay, Western Blot, Inhibition, Binding Assay