Mouse anti dsrna monoclonal antibody j2

Cell were lysed at 4 °C for 30 min with lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% [v/v] Triton X-100; protease inhibitor cocktail [Roche]). Cell lysates were centrifuged at 12,000 × g for 10 min and clarified supernatants were collected. The total protein concentrations were determined by the Bradford method against a bovine serum albumin standard. Protein was boiled in SDS-PAGE sample buffer (125 mM Tris–HCl, pH 6.8; 4% [v/v] SDS; 20% [v/v] glycerol; 0.004% [w/v] bromphenol blue), and separated by 10% SDS-PAGE. Gels were electro-blotted onto a polyvinylidene fluoride membrane (Pall) using a semi-dry transfer system (Bio-Rad). DENV-2 E and CA proteins were detected with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV C polyclonal antibody (Novusbio), respectively. DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS). Endogenous β-tubulin was detected with a mouse anti-β-tubulin monoclonal antibody (Sigma Aldrich). Primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP)-linked antibodies or anti-mouse IgG HRP-linked antibodies (Cell Signaling Technology).

Cells were grown on glass coverslips, fixed in 4% (w/v) paraformaldehyde at room temperature for 10 min and quenched with 50 mM NH₄Cl for 5 min. Cells were then permeabilized with 0.1% (v/v) Triton X-100 for 15 min and blocked in 10% (v/v) normal goat serum (Sigma Aldrich) for 15 min. DENV-2 NS3 protein was detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich). DENV-2 E and C were probed with a rabbit anti-DENV E polyclonal antibody (GeneTex) and a rabbit anti-DENV C polyclonal antibody (Novusbio), respectively. Double strand (ds) RNA was probed with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS). The secondary antibodies were Alexa Fluor 647-conjugated goat anti-rabbit (Thermo Fisher Scientific) or Cy5-conjugated goat anti-mouse (Life Technologies). Nuclei were stained with 1 µM 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). The coverslips were mounted onto slides with ProLong Gold antifade reagent (Life Technologies). Fluorescent images were captured using a Zeiss 780 NLO confocal scanning microscope (Zeiss) with 63× objective lenses and standard lasers and filters for Alexa Fluor 647, Cy5, and DAPI fluorescence.