Alexafluor594 conjugated cholera toxin subunit b

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.

Mouse monoclonal antibody against EV71 capsid protein VP-1 was purchased from Abcam (Cambridge, UK). Rabbit monoclonal antibody to AP2M1 (Abcam), Rabbit polyclonal antibodies to FCHO2 (ProSci, Poway, CA, USA), eIF4G (Cell Signaling Technology, Danvers, MA, USA) and Enterovirus 71 2C and 3C (GeneTex, Irvine, CA, USA), Mouse monoclonal antibodies against clathrin (CLTC, Santa Cruz Biotechnology, Dallas, Texas, USA) and dynamin (Santa Cruz Biotechnology) were used in Western-blot detection of the respective proteins. The secondary antibody conjugated to Allophycocyanin (APC) was from BD Biosciences (Franklin Lakes, CA, USA). AlexaFluor594-Conjugated AffiniPure Goat Anti-Mouse IgG (H + L) was from ZSGB-BIO (Beijing, China). Fluoroshield Mounting Medium with DAPI was from Abcam. AlexaFluor 488 conjugated transferrin from human serum and AlexaFluor 594 conjugated cholera toxin subunit B were from ThermoFisher scientific (Waltham, MA, USA). Chloropromazin and dynasore were purchased from Selleckchem (Shanghai, China). Guanidine hydrochloride (GnHCl) was from MedChem Express (Monmouth Junction, NJ, USA). All drugs were prepared according to the manufacturer’s instructions under sterile conditions.