Flash edt2

The cellular extracts of virus‐infected MDCK cells were prepared as described in previous section of immunoblotting. Before FlAsH‐EDT₂ (Invitrogen) was added, the extracts were treated with or without β‐mercaptoethanol (final concentration 35.8 mm; Merck) and boiled for 5 min. To test FlAsH‐EDT₂ binding ability on the native NS1‐tc proteins, the extract was mixed with FlAsH‐EDT₂ without any treatment. The labeling condition was set at 70 °C for 10 min with FlAsH‐EDT₂ (final concentration 2 μm) and then further incubated at room temperature for 1 h. Finally, the extract was analyzed by SDS/PAGE on a 12% polyacrylamide gel. The protein/FlAsH complexes were visualized on the gel with an appropriate filter for FlAsH fluorescence (excitation at 480 nm and emission at 535 nm) in Kodak image station 2000MM system. The same gel could be further analyzed for the total protein by Coomassie Blue staining or the specific NS1 with anti‐NS1 antiserum on the immunoblot.

The protocol for the association of FlAsH-EDT₂ with β-tubulin-TC in cell was adapted from the previous study (Hoffmann et al., 2010 (link)) so as to maximize the labeling fraction while maintaining cell viability. The engineered U2OS cells expressing β-tubulin-TC were grown to 80~90% confluency in a 30 mm cell culture dish, and then were gently washed with Opti-MEM (Thermo Fisher) twice, and then stained in 2 ml Opti-MEM with 1 μM FlAsH-EDT₂ (Thermo Fisher) for 2 hr. To reduce the non-specific binding of FlAsH, the stained cells were subsequently incubated in Opti-MEM containing 250 μM 1,2-Ethanedithiol (EDT, Alfa Aesar) for 10 min, followed by a gentle wash with Opti-MEM. The cells were incubated in DMEM with 10% FBS for 6~10 hr before imaging, because they were found to be interphase-arrested for the first ~5 hr after the incubation with 250 μM EDT. Every buffers and media above were pre-warmed at 37°C before use. All incubation steps were performed at 37°C in a humidified atmosphere with 5% CO₂.

All chemicals, unless explicitly stated, were purchased from Sigma Aldrich. Immunocytochemistry PBS (iPBS) (137 mM NaCl, 2.7 mM KCl, 10 mM NaH₂PO₄, 1.8 mM KH₂PO₄, 1 mM CaCl₂ and 0.5 mM MgCl₂, pH 7.40, sterile filtered) was used for washing of fixed cells. FlAsH-EDT2 was purchased from ThermoFischer Scientific.

HL-1 cardiomyocytes infected with the various CD36 constructs were cultured on gelatin/fibronectin-coated coverslips in 24-well plate. Prior to labelling, cells were incubated in serum-free depletion medium for 16 h. Next day, medium was removed and cells were washed twice with HBSS with 5.6 mM glucose. Cells were incubated with 500 nM FlAsH-EDT₂ (Thermofisher) in HBSS/glucose medium for 1 h at 37°C. After the aspiration of FlAsH-EDT₂, non-specifically bound dye was removed by incubation with 250 μM EDT for 10 min at 37°C. Cells were washed seven times with HBSS/glucose and fixed with 4% formaldehyde afterwards for 10 min at room temperature. Subsequently, cells were rinsed twice in PBS and permeabilised by 0.2% Triton X-100 in PBS for 15 min at room temperature, followed by 90 min of incubation with anti-Myc antibody (Cell Signaling Technology; 1:4000), anti-v-ATPase a2 (Abcam; 1:100), or anti-v-ATPase B2 (Abcam; 1:1000) in blocking buffer (5% FCS in 0.02% Triton X-PBS) and Texas Red conjugated goat anti-mouse or rabbit antibody (SouthernBiotech; 1:100) in blocking buffer for 1 h at room temperature in darkness. Coverslips were washed and mounted with DABCO-glycerol medium (Sigma-Aldrich) containing DAPI (1:10,000; Sigma-Aldrich).