Rnt sci 10010200

Mock or SINV WT- infected cells were plated on 8-well LabTek slide (Merck Millipore), were fixed with 4% formaldehyde (Merck) diluted in PBS 1X for 10 min at room temperature and then washed 3 times with PBS 1X. Cells were blocked in blocking buffer (0.1% Triton X-100; PBS 1 X; 5% normal goat serum) for 1 h. The following primary antibodies were diluted 1:400 in blocking buffer and incubated over night at 4 °C: mouse anti-dsRNA J2 (RNT-SCI-10010200; Jena bioscience), mouse anti-DDX5 (67025 Proteintech) or rabbit anti-DDX5 (ab21696; Abcam), anti-DDX17 (mouse, sc-27112; Santa Cruz Biotechnology), anti-capsid (rabbit, kind gift of Diane Griffin). Cells were washed with PBS 1X-Triton 0.1%. and incubated for 1 h at room temperature with goat anti-mouse Alexa 594 (A11032, Invitrogen) or goat anti-rabbit Alexa 488 (A11008, Invitrogen) secondary antibodiesdiluted at 1:1000 in PBS 1X-Triton X-100 0.1%. DAPI staining was performed for 5 min in PBS 1X to reveal the nuclei (D1306, Invitrogen, Thermo Fisher Scientific). Slides were mounted on coverslips with Fluoromount-G mounting media (Invitrogen, Thermo Fisher Scientific) and observed by confocal microscopy (LSM780, Zeiss) with a 40X or 63X objective. Images were analysed using Image J software and fluorescence intensity profiles were obtained.

mRNA samples were spotted onto the Amersham Hybond-N⁺ membrane and air-dried. After being blocked in TBT-T [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05%(v/v) Tween-20] containing 5% (w/v) skim milk (Wako) for 1 h at room temperature, the blot was incubated with anti-dsRNA clone J2 (Sigma-Aldrich, MABE1134-100UL or Jena Bioscience, RNT-SCI-10010200) for 1 h at room temperature, which 1000-fold diluted with TBS-T containing 0.5% skim milk. After being washed with TBS-T, the blot was further incubated with anti-mouse IgG-HRP (Sigma-Aldrich, A9044) for 1 h at room temperature, diluted by 5000-fold with TBS-T containing 0.5% skim milk. After washing with TBS-T, the blot was incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo) and visualized on a ChemiDoc Touch MP imaging system (Bio-Rad). siRNA Ladder Marker (Takara, cat# 3430) was used as a positive control of dsRNA.

Cells were fixed in 4% (v/v) paraformaldehyde (PFA) in PBS at 37 °C for 30 min, permeabilized with PBS containing 0.1% (w/v) Triton-X and blocked with 3% (v/v) BSA in PBS at RT for 30 min. Adipocyte cytoplasm was stained with wheat germ agglutinin conjugated with Alexa Fluor^® 555 (W32464, Thermo Fisher Scientific) according to the manufacturer’s instruction before fixation. Adipocytes and macrophages were both incubated with mouse anti-dsRNA monoclonal antibody J2 (1:500; RNT-SCI-10010200, Jena Bioscience, Jena, Germany) or rabbit anti-dsRNA monoclonal antibody J2 (1:500, Kf-Ab01299-23.0, kerafast, Boston, MA, USA) to detect viral RNA. Additionally, macrophages were stained with mouse anti-CD68 monoclonal antibody (1:200; 14-0688-82, Invitrogen). Both primary antibodies were diluted in 3% (v/v) BSA in PBS incubated at 4 °C overnight. Secondary antibodies Alexa Fluor^® 488-conjugated donkey anti-rabbit (711-545-152, Jackson ImmunoResearch) and Cy-3-conjugated donkey anti-mouse (715-165-150, Jackson ImmunoResearch) were each applied at 1:500 dilution. Cells were mounted with DAPI Fluoromount-G (Southern Biotech) and examined with an AxioObserver Z.1 microscope (Carl Zeiss AG).