inverted fluorescence microscope nikon eclipse t i 2-e  (Nikon)

Journal: bioRxiv

Article Title: Viperin binds STING and enhances the type-I interferon response following dsDNA detection

doi: 10.1101/493098

Figure Lengend Snippet: (A) HeLa cells were transfected with a viperin-flag construct 24 hrs prior to stimulation with poly dA:dT for 2 hrs and probing with mouse monoclonal anti-flag (Sigma) and rabbit monoclonal anti-STING (Millipore) or anti-TBK1 (Cell Signalling) antibodies, then subject to Duolink® In Situ Red Mouse/Rabbit PLA and DAPI staining. Imaged on Nikon Eclipse T i -E fluorescence inverted microscope. Scale bar represents 150 µm. Original magnification is X20. (B) Huh-7 or (C) HeLa cells were transfected with viperin-flag and either (B) TBK1-myc or (C) STING-myc constructs 24 hrs prior to stimulation with poly dA:dT for 2 hrs and immunofluorescence staining with rabbit monoclonal anti-flag (Sigma) and mouse monoclonal anti-myc (Millipore) antibodies followed by an Alexa555-conjugated goat anti-mouse (Invitrogen) and Alexa488-conjugated goat anti-rabbit (Invitrogen) secondary, as well as DAPI and (C) BODIPY staining. Imaged on Ziess Confocal LSM 780 microscope. Scale bar represents 15 µm. Original magnification is X63. (D and E) Huh-7 cells were transfected with viperin-mCherry or control-mCherry and either (D) TBK1-myc or (E) STING-myc constructs 24 hrs prior to stimulation with poly dA:dT for 2 hrs, and cell extracts were immunoprecipitated with rabbit monoclonal anti-mCherry antibody (Biovision) and subject to immunoblot analysis with indicated antibodies. (D) Cell extract was subject to DSS crosslinking prior to lysis and immunoprecipitation. Immunoblots are representative of at least 2 independent experiments.

Article Snippet: Imaged on Nikon Eclipse T i -E fluorescence inverted microscope.

Techniques: Transfection, Construct, In Situ, Staining, Fluorescence, Inverted Microscopy, Immunofluorescence, Microscopy, Immunoprecipitation, Western Blot, Lysis

Journal: bioRxiv

Article Title: Viperin binds STING and enhances the type-I interferon response following dsDNA detection

doi: 10.1101/493098

Figure Lengend Snippet: (A and B) HEK293T cells were transfected with viperin-mCherry, STING-myc and either K63-Ub-HA, K27-Ub-HA or wt-Ub-HA constructs 24 hrs prior to (A) visualisation by fluorescence microscopy, after which (B) cell extracts were immunoprecipitated with mouse monoclonal anti-myc antibody (Millipore) and subject to immunoblot analysis with indicated antibodies. Imaged on Nikon Eclipse T i -E fluorescence inverted microscope. Scale bar represents 200 µm. Original magnification is X20. (C) HEK293T cells were transfected with combinations of viperin-flag, STING-myc and TBK1-myc constructs 24 hrs prior to immunoblot analysis with indicated antibodies. (D and E) Huh-7 cells were transfected with viperin-GFP, TBK1-mCherry and K63-Ub-HA constructs 24 hrs prior to (D) visualisation by fluorescence microscopy, and (E) stimulation with poly dA:dT for 2 hrs, after which cell extracts were immunoprecipitated with rabbit monoclonal anti-mCherry antibody (Biovision) and subject to immunoblot analysis with indicated antibodies. Imaged on Nikon Eclipse T i -E fluorescence inverted microscope. Scale bar represents 100 µm. Original magnification is X20. (F) Wild-type and viperin −/− primary MEFs were stimulated with poly dA:dT for 2 hrs, and cell extracts were immunoprecipitated with mouse monoclonal anti-K63-Ub antibody (Enzo) and subject to immunoblot with indicated antibodies. Immunoblots are representative of at least 2 independent experiments.

Article Snippet: Imaged on Nikon Eclipse T i -E fluorescence inverted microscope.

Techniques: Transfection, Construct, Fluorescence, Microscopy, Immunoprecipitation, Western Blot, Inverted Microscopy