FIG 3.

Detection of Env-Ser5 interaction by BiFC. (A) 293T cells were transfected with 1 μg Ser2-VN, Ser2-VC, Ser5-VN, and Ser5-VC expression vectors, and their expression was determined by Western blotting using anti-HA and anti-FLAG. (B) Indicated amounts of Ser2-VN, Ser2-VC, Ser5-VN, and Ser5-VC expression vectors were transfected into 293T cells. After permeabilization, Ser2- and Ser5-VN were stained with Pacific Blue-conjugated anti-HA, and Ser2-VC and Ser5-VC were stained with APC-conjugated anti-FLAG. Fluorescent signals were detected by flow cytometry. (C) Indicated amounts of Ser2, Ser5, and NL Env BiFC fusion expression vectors were transfected into 293T cells individually or pairwise. Their BiFC fluorescent green signals were analyzed by flow cytometry. The levels of Env interaction with Ser2 and Ser5 were then calculated and presented as relative values, with the levels of the Ser5-VN/NL-VC pair set as 100%. The levels of Ser2-VN, Ser2-VC, Ser5-VN, and Ser5-VC expression were determined by Western blotting. Error bars represent SDs of the results from three independent experiments. (D) Indicated Ser5-Env BiFC pairs were expressed in 293T cells. Cells were stained with Alexa Fluor-647-conjugated anti-HA or Pacific Blue-conjugated anti-FLAG. Fluorescent signals were visualized by confocal microscopy (scale bar = 5 μm). (E) Indicated Env-Env and Ser5-Env BiFC pairs were expressed with a mCherry fusion protein expressing the N-terminal 14 residues of the ER protein calnexin (CNX). Their colocalization was determined by confocal microscopy (scale bar = 5 μm). DAPI, 4′,6-diamidino-2-phenylindole.