FIG 12.

The ANDV-NSs protein and MAVS interact in Huh-7 cells. (A) Huh-7 cells were transfected with plasmids encoding HA-ANDV-NSs and FLAG-MAVS. At 24 h p.t., cells were fixed with 4% PFA and permeabilized using PBS-Triton X-100. ANDV-NSs and MAVS were detected by immunofluorescence (IF) using rabbit anti-HA and mouse anti-FLAG primary antibodies in combination with Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 594 donkey anti-mouse secondary antibodies (upper panels). The inset in the upper right corner of the upper right panel shows mock-transfected cells as a negative control for IF. PLA secondary antibodies were used following the manufacturer’s instructions (bottom panels). The inset in the upper left corner of the bottom right panel shows transfected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of the PLA reaction. Vectashield with DAPI was used as a mounting medium. The images were obtained on an Olympus epifluorescence microscope and processed with ImageJ. (B) Huh-7 cells were transfected with a plasmid encoding HA-ANDV-NSs in combination with FLAG-EV. At 24 h p.t., cells were fixed with 4% PFA and permeabilized using PBS-Triton X-100. The ANDV-NSs protein and endogenous MAVS were detected by IF using rabbit anti-HA and mouse anti-MAVS primary antibodies and Alexa Fluor 488 donkey anti-rabbit and Alexa Fluor 594 donkey-anti-mouse as secondary antibodies. The inset in the upper right corner of the upper right panel shows mock-transfected cells as a negative control for IF. PLA secondary antibodies were used as indicated above. The inset in the upper right corner of the bottom right panel shows transfected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of the PLA reaction. Vectashield with DAPI was used as a mounting medium. The images were obtained on an Olympus epifluorescence microscope and processed with ImageJ.