Mouse anti dsrna antibody

Lipofectamine 3000 transfection reagent was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Fugene HD transfection reagent was from Promega (Madison, WI, USA). Mouse anti-GFP antibody was from Santa Cruz Biotechnology (Dallas, Texas, USA). Mouse anti-dsRNA antibody was from SCICONS (J2 mAb, Budapest, Hungary). Alexa Fluor 594-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) was purchased from ZSGB-BIO (Beijing, China). Fluoroshield Mounting Medium with DAPI was from Abcam (Cambridge, UK). Luciferase assay system was from Promega. Micrococcal nuclease was from New England BioLabs (Ipswich, MA, USA).

In generating rabbit anti-nsP1 serum, a 911-bp NcoI–SmaI fragment (AF369024, 637–1548) of partial nsp1 gene from the pFastBac1-VSVG-CHIKV replicon-GFP was subcloned into the restriction enzyme sites of pET32c. The expression, purification, and rabbit immunization of recombinant nsp1 were performed in accordance with methods described in a previous report^{57 (link)}. AP-61 cells were transduced using WT/eGFP at an MOI of 5. At 4 dpt, cells were fixed and incubated using the above-mentioned rabbit anti-nsp1 serum (1:100) or Mab anti-GFP antibodies (Santa Cruz Biotechnology, Inc.) (1:100) and mouse anti-dsRNA antibody (SCICONS) for 1 h. Cells were subsequently washed three times using PBS and incubated with either Alexa Fluor 594-conjugated secondary antibodies or Alexa Fluor 488-conjugated secondary antibodies as well as Hoechst 33342 (10 µM) for 1 h. After a final wash using PBS, images of cells were captured using an inverted fluorescence microscope (DMi8 Microscope, Leica Microsystems).