In ANDV-infected Huh-7 cells, hMex3A interacts with eIF4G, but not with the ANDV-N protein.
(A) Huh-7 cells were infected with ANDV (MOI 1), and 24 hpi transfected with a plasmid encoding for hMex3A-HA protein. Twenty-four hpt cells were fixed (PFA 4%) and permeabilized (PBS-Triton). The ANDV-N and hMex3A-HA proteins were detected using a mouse monoclonal anti-ANDV-N or a rabbit polyclonal anti-HA as primary antibodies. For the IF, a donkey anti-mouse Alexa 488 or a donkey anti-rabbit Alexa 594 were used as the secondary antibody. For the hMex3A-HA/ANDV-N interaction assay, PLA secondary antibodies were used following the manufacturer’s instructions. (B-D) Huh-7 cells were infected with ANDV (MOI 1), and 24 hpi transfected with a plasmid encoding for hMex3A-HA protein. Twenty-four hpt cells were fixed (PFA 4%) and permeabilized (PBS-Triton). (B) The expression of the ANDV-N and eIF4G was confirmed by IF using a mouse monoclonal anti-ANDV-N and a polyclonal anti-eIF4G as primary antibodies and a donkey anti-mouse Alexa 594 and donkey anti-rabbit Alexa 488 as secondary antibodies, respectively. (C) Endogenous eIF4G and recombinant hMex3A-HA protein were detected by IF using a rabbit polyclonal anti-eIF4G antibody and a monoclonal mouse anti-HA antibody as primary, and an Alexa 594 donkey anti-rabbit or a donkey anti-mouse Alexa 488 as the secondary antibodies. 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 antibodies. (D) ANDV-infected cells were incubated with primary antibodies against hMex3A-HA and eIF4G as in (C), but PLA secondary antibodies were used following the manufacturer’s instructions. In the upper right corner of the merge, the inset shows infected and transfected cells without primary antibodies but with the PLA secondary antibodies added as a negative control of PLA. (A-D) Vectashield with DAPI was used as mounting media. The images were obtained in Olympus epifluorescence Microscope and processed by Image J.
