Figure 1.

Subcellular localization and expression kinetics of ASFV CD2v. (A) PK15 cells were mock transfected or transfected with pCD2v-HA, fixed at various times pt, incubated with anti-HA primary antibody, sequentially stained with Alexa fluor 488-labeled secondary antibody and DAPI and examined by confocal microscopy. (B) CD2v-HA-expressing PK15 cells hemadsorbed swine red blood cells (RBCs). PK15 cells were transfected with plasmid pCD2v-HA for 24 h, incubated with swine RBCs overnight and examined using light microscopy (×100). CD2v-dependent rosetting is indicated by the arrowheads. (C) PK15 cells mock transfected or transfected with pCD2v-HA were harvested at the indicated times pt and total cell protein extracts were resolved by SDS-PAGE, blotted and incubated with antibodies against HA. (D) Detection of CD2v in the culture supernatant. PK15 cells were transfected as above and supernatants harvested at various times pt. Cleared supernatants (50 μL) were resolved by SDS-PAGE and analyzed by Western blot using antibodies against HA. * denotes the non-specific background band due to serum proteins in the supernatant. (E) Primary swine macrophages were mock transfected or transfected with a pCD2v-HA, fixed at 24 h pt, incubated with anti-HA primary antibody, sequentially stained with Alexa fluor 594-labeled secondary antibody and DAPI and examined by confocal microscopy. (F) Detection of CD2v in the culture supernatant of primary swine macrophages transiently expressing CD2v. Cells were transfected as above, incubated for 24 h and supernatants were immunoprecipitated with anti-HA antibody, resolved in SDS-PAGE and probed with the anti-HA antibody. Results for (A–F) are representative of two independent experiments.