FIG 6.

Distinct cellular localization patterns of BILF1 receptors. (A) HEK-293 cells were cotransfected with EBV BILF1, PtroLCV1 BILF1, PpygLCV1 BILF1, and SsynLCV1 BILF1 and the farnesylated enhanced green fluorescent protein (eGFPF). The representative pictures show the localization of the BILF1 receptors detected with a primary HA antibody against the HA tag expressed at the N termini of the receptors (red signal, left side), the signal of eGFPF (green signal, middle), and the merge signals of the BILF1 receptors with eGFPF (orange signal, right side). (B) Representative pictures of EBV BILF1- and PpygLCV1 BILF1-expressing HEK-293 cells (top) and SsynLCV1 BILF1-expressing HEK-293 cells (bottom), cotransfected with PtroLCV1 BILF1. EBV, PpygLCV1, and SsynLCV1 BILF1 molecules were detected with an anti-HA FITC-conjugated antibody using an N-terminal HA tag (green signal). PtroLCV1-BILF1was detected with a primary antibody against the c-myc tag (N terminal) and a secondary Cy5-conjugated antibody (red signal). (C) The graph shows cell surface expression levels of the BILF1 receptors as estimated in ELISA using an N-terminal HA tag (means ± SEM; n = 3). (D) Representative pictures of the constitutive internalization of the BILF1 receptors determined by antibody uptake studies. HEK-293 cells were transfected with HA-tagged EBV BILF1, PtroLCV1 BILF1, PpygLCV1 BILF1, and SsynLCV1 BILF1. Forty-eight hours after the transfection, receptors present at the cell surface were stained with a hemagglutinin antibody for 1 h at 4°C. Subsequently, cells were incubated at 37°C for 30 min to induce internalization and fixed afterwards. Labeled receptors still residing at the cell surface were detected with a FITC-conjugated antibody (green signal) prior to permeabilization, while internalized receptors were detected with a rhodamine-conjugated antibody (red signal).