Tetherin inhibits the release of PFV. (A, B) 293T cells were transfected with PFV infectious clone pcPFV along with human tetherin expression plasmids (HuTHN). At 44-48 h after transfection, the amount of virus in supernatants was analyzed by infecting an indicator cell line PFVL, Western blotting (A), and viral DNA copy number assay (B). (C, D) 293T cells were transfected with pcPFV, HuTHN, and HIV-1 Vpu expression plasmid. The virus titer in the supernatants was analyzed by infecting PFVL. The luciferase activity and viral DNA copy number were normalized to the samples transfected with empty vectors. The cell lysates were immunoblotted with anti-Gag antibody. The same blots were reprobed with anti- β-actin antibody and anti-FLAG antibody. The viral particles were immunoblotted with anti-Gag antibody and Odyssey goat anti-rabbit IRDye 680. The intensity showed below the blots was performed with LI-COR analysis software.