stat1 expressing plasmid  (Addgene inc)

Journal: Scientific Reports

Article Title: Amelioration of Experimental autoimmune encephalomyelitis and DSS induced colitis by NTG-A-009 through the inhibition of Th1 and Th17 cells differentiation

doi: 10.1038/s41598-018-26088-y

Figure Lengend Snippet: NTG-A-009 reduces Th1 and Th17 cells differentiation through the inhibition of JAK/STAT pathway. ( a ) Naïve CD4 + T cells isolated from spleen and lympnodes were cultured with or without NTG-A-009 under Th1 condition and re stimulated with anti-CD3 (1 μg/ml) and anti-CD28(1 μg/ml) followed by the analysis of the expression of indicated proteins in JAK/STAT pathway by western blot. Phosphorylated and total form of STAT1, STAT4, and JAK1/2 were analyzed by immunoblotting under Th1 condition. ( b ) Phosphorylated and total form of STAT3 and JAK1/2 were detected by immunoblotting under Th17 condition. Full length blots and gels were shown in supplementary information- 7, 8, 9 and 10. Data are the representative of three independent experiments.

Article Snippet: Cells were then transfected with an STAT3 (pcDNA3-STAT3-Y705F, addgene plasmid) and STAT1 expressing plasmid (STAT1 alpha Y701F pRc/CMV, addgene plasmid) using fugene HD transfection reagent (Promega Madison, USA) as described by manufacturer protocol.

Techniques: Inhibition, Isolation, Cell Culture, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibition of p38 Mitogen-activated Protein Kinase Impairs Influenza Virus-induced Primary and Secondary Host Gene Responses and Protects Mice from Lethal H5N1 Infection

doi: 10.1074/jbc.m113.469239

Figure Lengend Snippet: FIGURE 4. p38 MAPK inhibition affects H5N1-induced IFN expression. A, impact of p38 MAPK inhibition on the IFN promoter activity. Vero cells were transfected with the IFN promoter for 24 h. Cells were preincubated with 20 M SB 202190 or left untreated and subsequently stimulated with 500 ng of total RNA isolated from infected A549 cells (8 h, 5 m.o.i.). Total RNA from uninfected A549 cells was used as control. 5 h p.s. promoter activity was measured by a luciferase assay and the results are depicted as mean n-fold ( S.D.) of three independent experiments normalized to controls. B, Western blot analysis of total lysates of HUVEC treated with UV-inactivated, filtered conditioned media from mock-infected control cells (lanes 5 and 7) and KAN-1-infected cells (5 m.o.i., 5 h) (lanes 6 and 8). Donor cells were pretreated with DMSO (lanes 1 and 3) or SB 202190 (20 M, lanes 2 and 4). STAT1 Tyr701 phosphorylation was detected 15 min aftertreatmentwithconditionedmedium(upperpanel).EqualloadingwasverifiedbythedetectionoftotalERK2(lowerpanel).Blotsarerepresentativeofthree independent experiments.

Article Snippet: The expression plasmid pRC/CMV STAT1 Y701F was obtained from Addgene, provided by J. Darnell (Laboratory of Molecular Cell Biology, Rockefeller University, New York) and previously described (24).

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Isolation, Infection, Control, Luciferase, Western Blot, Phospho-proteomics