py 701 stat1  (Cell Signaling Technology Inc)

Journal: Journal of Virology

Article Title: The Methyltransferase-Like Domain of Chikungunya Virus nsP2 Inhibits the Interferon Response by Promoting the Nuclear Export of STAT1

doi: 10.1128/JVI.01008-18

Figure Lengend Snippet: Interaction of STAT1 with the importin complex is unaffected in the presence of CHIKV nsP2. HEK293 cells were mock transfected or transfected with a plasmid expressing a FLAG-tagged human importin-α5 (impα5) in combination with a plasmid expressing mCherry (mock), Ebola virus VP24 (positive control), or one of the mCherry-2A-nsP2 constructs. Whole-cell lysate was loaded on SDS-PAGE and stained for FLAG and CHIKV nsP2. The coimmunoprecipitation (Co-IP) product was stained for FLAG and STAT1. The numbers at the bottom indicate the importin-α5/STAT1 ratios.

Article Snippet: The cells were washed once in stain buffer (BD Pharmingen, San Diego, CA), followed by incubation with anti-pY(701)-STAT1 conjugated to peridinin chlorophyll protein (PerCP)-Cy5.5 (BD Pharmingen) or anti-IFNAR 4G8 monoclonal antibody (MAb) for 45 min at room temperature in the dark.

Techniques: Transfection, Plasmid Preparation, Expressing, Positive Control, Construct, SDS Page, Staining, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: The Methyltransferase-Like Domain of Chikungunya Virus nsP2 Inhibits the Interferon Response by Promoting the Nuclear Export of STAT1

doi: 10.1128/JVI.01008-18

Figure Lengend Snippet: nsP2 inhibits JAK/STAT inhibition by inducing the nuclear export of pSTAT1. Blocking nuclear export indicates the functional nuclear import of STAT1. Vero cells were transfected with either mCherry-2A-nsP1, mCherry-2A-nsP2, or one of its mutants; 16 hpt, the cells were mock treated or treated with the nuclear exportin CRM1 blocker LMB for 3 h prior to 30-min stimulation with IFN-β. The cells were fixed, permeabilized, and stained for pSTAT1. The bars represent the means from 3 independent experiments, and the error bars indicate SEM. The asterisks indicate significant differences as a result of LMB treatment (Student's t test; P < 0.05).

Article Snippet: The cells were washed once in stain buffer (BD Pharmingen, San Diego, CA), followed by incubation with anti-pY(701)-STAT1 conjugated to peridinin chlorophyll protein (PerCP)-Cy5.5 (BD Pharmingen) or anti-IFNAR 4G8 monoclonal antibody (MAb) for 45 min at room temperature in the dark.

Techniques: Inhibition, Blocking Assay, Functional Assay, Transfection, Staining

Journal: Journal of Virology

Article Title: The Methyltransferase-Like Domain of Chikungunya Virus nsP2 Inhibits the Interferon Response by Promoting the Nuclear Export of STAT1

doi: 10.1128/JVI.01008-18

Figure Lengend Snippet: The nsP2 C-terminal MTase-like domain promotes CRM1-mediated export of STAT1. (A) Vero E6 cells expressing the indicated nsP2 variants were treated with LMB for 3 h or left untreated prior to 30 min of IFN-β stimulation. The cells were fixed and stained for pSTAT1. Nuclear pSTAT1 was analyzed for cells that expressed nsP2, and the LMB-treated samples were normalized to their untreated controls. The results are depicted as fold change (LMB treated over untreated). The bars represent means ± SEM from three independent experiments. The asterisks indicate significant increases in nuclear pSTAT1 induced by LMB treatment (Student's t test; P < 0.05). (B) In the presence of CHIKV nsP2, STAT1/2 dimers interact effectively with importin-α5 and are transported across the nuclear pore. In the nucleus, the C-terminal domain of nsP2 promotes CRM1-mediated nuclear export of STAT1 (indicated by arrows and plus sign), inhibiting the JAK/STAT signaling pathway and preventing induction of ISG expression (indicated by the dashed arrow and minus sign).

Article Snippet: The cells were washed once in stain buffer (BD Pharmingen, San Diego, CA), followed by incubation with anti-pY(701)-STAT1 conjugated to peridinin chlorophyll protein (PerCP)-Cy5.5 (BD Pharmingen) or anti-IFNAR 4G8 monoclonal antibody (MAb) for 45 min at room temperature in the dark.

Techniques: Expressing, Staining