0.22 μm nitrocellulose membrane

The concentration of melittin peptides in the solution was determined by a UV–Vis spectrophotometer (PerkinElmer, United States). All buffers for peptide solutions were filtered through 0.22-μm nitrocellulose membranes before usage (Millipore). Melittin peptides were dissolved in 6 M guanidine-HCl solution to unfold peptides and measured the UV absorbance at the wavelength of 280 nm. The concentration of peptide solutions was calculated by Beer–Lambert law with the extinction coefficients of 5690 M⁻¹ cm⁻¹ for the tryptophan in the peptide sequence.

Cells seeded in 6-well plates were lysed using 500μl the mammalian protein extraction reagent RIPA (Beyotime, Beijing, China), together with 1μl protease inhibitor cocktail (Roche, Pleasanton, CA). After centrifugation at 4°C for 30 min at 18,000 g, the supernatant was separated and stored on ice. 40μg protein lysates were separated by 12% SDS-PAGE, transferred to 0.22-μm nitrocellulose membranes (Millipore, Boston, MA). The membranes were blocked with 5% fat-free milk for 3 h at room temperature, followed by incubation with primary antibodies against p53 and MDM2 (Santa Cruz, Dallas, TX) at 4°C overnight. An anti-GAPDH antibody (CWBiotech, Beijing, China) was used as a control. Then the membranes were washed five times and incubated with HRP-conjugated secondary antibody (CWBiotech, Beijing, China) for 2 h at room temperature. Signals were visualized using the ECL chromogenic substrate (Thermo scientific, Rockford, IL) and quantified by densitometry using the Quantity One software (Bio-Rad, Berkeley, CA).

To start the transport assay, loaded proteoliposomes (674 μg lipid) were diluted into Reaction Buffer containing 20 mM Tris/HEPES, pH 7.5, 1 μM valinomycin, and NaCl/KCl/ChCl concentrations varied depending on the experiment (see Supplementary file 1 for details). No additional radiolabeled substrate was added to the Reaction Buffer, so diluting the isotope-loaded proteoliposomes (which will have significant extra-liposomal radiolabeled substrate present) into the Reaction Buffer was sufficient to set the desired substrate gradient. The extent of proteoliposome dilution was therefore dictated by the succinate gradient we wished to achieve. Samples were taken at the specified timepoints, rapidly filtered on 0.22-μm nitrocellulose membranes (Merck Millipore) to collect the proteoliposomes, then washed by addition of 4 ml of ice-cold Quench Buffer (20 mM Tris/HEPES, ChCl osmotically balanced to inside buffer). The filters were dried, dissolved in liquid scintillation cocktail (FilterCount, PerkinElmer), and counted on a Trilux β counter (PerkinElmer). The initial point (t = 0) value was determined by diluting preloaded proteoliposomes directly into ice-cold 2 ml Quench Buffer, rapidly filtering and washing with 4 ml of Quench Buffer. No initial point was taken for vSGLT; instead a 5 s time-point was taken to act as the initial value.

Infected and mock U937 macrophage cell supernatants were collected at 120 h p.i. and centrifuged 10 min at 300 × g to pellet cell debris. The supernatants were centrifuged again at 2,000 × g for 20 min at 4°C, and the resulting supernatants were filtered through 0.22 μm nitrocellulose membranes (Merck Millipore). These eluates were concentrated using Amicon Ultra-15 MWCO 100K centrifugal concentrators (Merck Millipore) at 4,000 × g for 20 min at 4°C. The concentrated preparation was precipitated via ultracentrifugation (UC) at 100,000 × g for 60 min at 4°C using an Optima MAX-TL centrifuge (TLS-55 rotor -k Factor 100.2- Beckman Coulter), and the pellet was resuspended in 100 μL PBS and stored at −80°C until use. Pellets from DENV-infected macrophages was named P (+) and that from non-infected cells was named P (-).

Cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, China) supplemented with protease inhibitors cocktail (Roche, Switzerland) and PMSF (Roche, Switzerland). Protein concentration was measured with the Bio-Rad protein assay kit. Protein extractions of 50 μg were separated by 12% SDS/polyacrylamide gel electrophoresis (SDS/PAGE), followed by electro-transferring to 0.22 μm nitrocellulose membranes (Sigma–Aldrich, U.S.A.). Primary and secondary antibodies were sequentially added. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad). GAPDH was used as a loading control. GAPDH antibody was purchased from Sigma–Aldrich (U.S.A.), and cleaved-caspase-3 antibody was purchased from CST.