Journal: bioRxiv
Article Title: Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff
doi: 10.1101/2023.03.20.533404
Figure Lengend Snippet: (A) DF-1 cells were transfected with a plasmid coding Flag-tagged nsp15 (PXJ40F-nsp15). At 24 h.p.t, indirect immunofluorescence was performed with a chicken anti-Flag-tag antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV-WT or rIBV-nsp15-H238A at an MOI=1. At 18 h.p.i, indirect immunofluorescence was performed with a mouse anti-IBV-nsp15 monoclonal antibody (red), a rabbit anti-IBV-nsp12 polyclonal antibody (green) and the nuclei were stained with DAPI (blue). (C) Vero cells were infected with IBV or rIBV-nsp15-H238A with an MOI of 1. At 18 h.p.i., cells were treated with 100 μg/mL cycloheximide (CHX) for 15 min at 37°C and subjected to 7–47% sucrose density gradient ultracentrifugation (38,000 rpm for 3 h), and the fractions were analysed by Western blot to detect nsp15, Rsp6, eIF4E, and β-actin (left panel).
Article Snippet: Mouse anti-V5 (Thermo fisher scientific, #R961-25, horseradish peroxidase HRP-conjugated), mouse anti-HA (MBL, #M180-7, HRP-conjugated), mouse anti-Flag (MBL, #M185-7, HRP-conjugated), were diluted by 1:2500 for Western Blot; mouse anti-β-actin (CST, #3700S), rabbit anti-GFP (CST, #2956), chicken anti-Flag (Gentaur, #AFLAG), rabbit anti-phosphorylated PKR (Abcam, #ab32036), rabbit anti-PKR (CST, #12297), rabbit anti-phosphorylated eIF2α (CST, #3398), rabbit anti-eIF2α (CST, #5324), anti-RPS6 rabbit mAb (CST, #2217) and eIF4E rabbit mAb (CST, #2067), were diluted by 1:1000 dilution for Western Blot; rabbit anti-IBV N (provided by Prof Dingxiang Liu, South China Agricultural University, China) was diluted by 1:2000 for Western blot; rabbit anti-human PABPC1 (Abcam, #Ab21060, cross-reacts against chicken PABPC1 in DF-1 cells), rabbit anti-IBV-N, anti-IBV nsp12 rabbit pAb (provided by Prof Dingxiang Liu, South China Agricultural University, China), anti-IBV nsp15 mouse mAb (provided by Dr. Min Liao’s lab, Zhejiang University, China), were diluted by 1:500 for immunofluorescence; mouse anti-puromycin (Sigma-Aldrich, #MABE343) was diluted by 1:25000 for Western blot and 1:10000 for immunofluorescence; anti-dsRNA mouse mAb J2 (Scicons, #10010200) was diluted by 1:1000 for dot blot analysis.
Techniques: Transfection, Plasmid Preparation, Immunofluorescence, FLAG-tag, Staining, Infection, Western Blot
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