The ANDV-N protein and eIF4G interact in ANDV infected Huh-7 cells.

(A) Huh-7 cells were infected with ANDV (MOI 1), and 24 hours post-infection (hpi), cells were fixed (PFA 4%) and permeabilized (PBS-Triton). Expression of the ANDV-N and endogenous eIF4G proteins was confirmed by immunofluorescence (IF) using mouse monoclonal anti-ANDV-N or rabbit polyclonal anti-eIF4G as primary antibodies. As secondary antibodies, an Alexa 488 donkey anti-mouse or an Alexa 594 donkey anti-rabbit was used, respectively. As a negative control for the IF, the inset, in the upper right corner of the merge, shows ANDV infected cells where detection was conducted using only the secondary antibody. (B) ANDV-infected cells were incubated with primary antibodies as in (A), but PLA secondary antibodies were used following the manufacturer’s instructions. The inset, in the upper right corner of the merge, shows infected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of PLA. Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscope and processed by Image J. (C-D) HEK293T cells were cotransfected with the ANDV HA-N or HA-EV plasmids. Forty-eight hours later, cells were lysed, and immunoprecipitation (IP) assays were performed using protein A/G agarose coated with the corresponding antibody. The beads were washed and incubated with loading buffer at 95°C and the supernatant, which was used for western blotting. (C) Whole-cell lysate (WCL) of each sample and (D) IP fractions with the corresponding primary antibody highlighted on the right side of the panels. Western blotting was performed using mouse anti-HA, rabbit anti-eIF4G, and mouse anti-GAPDH. Horseradish Peroxidase (HRP)-conjugated Protein A/G was used to detect the primary antibodies.