Immunofluorescence Imaging of Transfected Cells

For immunofluorescence, HeLa or A549 cells were seeded onto glass cover slips 1 day before transfection. At 24 or 48 h post transfection, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. The cells were then stained with primary antibodies and Alexa Fluor-conjugated secondary antibodies. In the experiments using A3.01 T cells, transfected cells were cultured in round-bottom plates for 48 h. Cells were then fixed with 4% PFA and permeabilized with 0.2% saponin (except for surface GM1 staining), and stained with antibodies. For GM1 staining, fixed cells were stained with Alexafluor594-conjugated Cholera Toxin Subunit B (Thermo Fisher Scientific) for 30 min. Microscopic imaging was performed with an FV1000-D confocal laser scanning microscope (Olympus, Tokyo, Japan). Line plots of the fluorescence intensity were generated by ImageJ software (NIH, Bethesda, MD).
Quantitative analysis of subcellular localization of Gag–GFP was performed as previously reported34 (link)67 (link)68 (link). Briefly, HeLa cells expressing Gag–GFP were fixed and 20 random fields were inspected. Over 100 cells were analysed for the subcellular localization of Gag–GFP, which was either strongly evident at the PM only, at the PM with intracellular accumulations, or diffusely in the cytoplasm.