Qubit protein assay

Cell lysate was collected in 8 M urea with 2.5% SDS. Protein concentration was measured with Qubit Protein Assay (Thermo Fisher), with 30 μg of cell lysate and 15 μg of exosome protein loaded. Protein was denatured in 4× NuPAGE LDS sample buffer with 62.5 mM DTT for 10 min at 70 °C. Membranes were blocked with 5% milk in TBS with 0.1% Tween (TBST) and incubated with rabbit anti-SLC19A1 (Boster Biological Technology PB9504, 1:1000), rabbit anti-SLC38A2 (Sigma Aldrich SAB4502246, 1:1000), mouse anti-CD81 (Santa Cruz Biotechnology sc-166029, 1:1000), or rabbit anti-vinculin (Abcam ab129002, 1:1000) for 1 h at room temperature or overnight at 4 °C. Membranes were incubated in anti-rabbit HRP (Abcam ab16284, 1:1000) or anti-mouse HRP (R&D Systems HAF007, 1:1000) for 1 h at room temperature and developed using West-Q Pico ECL solution (GenDEPOT).

Cellular variants were seeded on 6-well plates and cultured in full medium to obtain 80% confluence, after which the medium was exchanged to starving. After 48 h (37 °C, 5% CO₂), the starvation medium was collected and centrifuged. The Qubit Protein Assay on a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Naarden, the Netherlands) was used to measure the protein concentration, and subsequently, 3 µg of proteins was used for gelatin-supplemented (2 mg/mL; Merck Life Sciences Sigma Aldrich, Darmstadt, Germany) SDS-PAGE electrophoresis. The gels were washed (2 × 30 min) with 2.5% Triton X-100 (Merck Life Sciences Sigma Aldrich, Darmstadt, Germany), followed by overnight incubation in a developing buffer (0.5 M Tris-HCl, 2 M NaCl, 50 mM CaCl₂, pH 7.5) at 37 °C. The Coomassie Brilliant Blue R-250 (Merck Life Sciences Sigma Aldrich, Darmstadt, Germany) was used to stain the resulting zymogram and the gel was subsequently washed with destaining solution (methanol:acetic acid:water, 3:1:6). Clear bands over a stained dark blue background appeared at areas of gel where gelatin was enzymatically degraded. The protein ladder was used to confirm the molecular weights of the Matrix Metalloproteinases (MMPs); their activity was estimated using ImageJ software [25 (link)]. The assay was performed in triplicate for each cellular variant.

Total protein concentration was used to normalize TNF-α and IL-2 concentrations by dividing TNF-α and IL-2 (pg/mL) by total protein concentration (mg/mL). Total protein concentration was determined using a Qubit Protein Assay (ThermoFisher, Waltham, MA, USA; Cat. No. Q33211). Samples were diluted 1:20 and the assay was performed as per manufacturer’s instructions. Samples were read on a microplate reader by diluting the three standards to 20 µg/mL, 15 µg/mL, 10 µg/mL, and 0 µg/mL. Fluorescence was read at excitation/emission maxima of 470/570 nm.

Approximately 200 μL of whole worms were lysed on ice by grinding in solubilization buffer (ground in aliquots of 100 μL gently pelleted worms per 350 μL buffer; 300 mm NaCl, 50 mm Tris–HCL pH 8, 10 mm MgCl₂, 1 mm EGTA, 200 μg mL⁻¹ heparin, 400 U mL⁻¹ RNAsin, 1 mm PMSF, 0.2 mg mL⁻¹ cycloheximide, 1% Triton X‐100, 0.1% sodium deoxycholate). After grinding, an additional 350 μL solubilization buffer was added (1050 μL total) and the lysates were incubated on ice for 30 min. Lysates were centrifuged at 12 000 × g at 4 °C for 10 min to collect debris. Protein content of the lysate supernatants was estimated using Qubit protein assay (Thermo Scientific, Waltham, MA, U.S.A.) and loaded equally onto 10–50% sucrose gradients in high salt resolving buffer (140 mm NaCl, 25 mm Tris–HCL pH 8, 10 mm MgCl₂). Gradients were resolved by ultracentrifugation in a Beckman SW41Ti rotor at 38 000 × g at 4 °C for 2 h. Fractions of the gradients were continuously monitored at absorbance of 254 nm using a Teledyne density gradient fractionator. The polysome fraction was collected and used for qRT–PCR as described above.

The concentrations of purified TOP3A variants were measured using a Qubit protein assay (Thermo Fisher Scientific). Reactions were carried out in a total volume of 20 μl in reaction buffer (25 mM Tris–HCl pH 7.4, 5 mM MgCl₂, 50 mM NaCl, 1 mM DTT and 100 μg/ml BSA) at 37°C for 30 min. Proteins were diluted using dilution buffer (20 mM tris‐Cl pH 8, 200 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 0.5 mM EDTA and 100 μg/ml BSA). In experiments involving two compound heterozygous variants, reactions contained 50 fmol of protein (for single variants) or 25 fmol for each of the two variants. The reaction products were then separated by denaturing PAGE as described above. Reaction products were electroblotted onto Hybond‐N+ membranes (Cytiva) at 50 V in 1 × TBE for 1 h. Membranes were crosslinked by exposure to 1200 mJ/cm² of 254 nm UV. Membranes were hybridised overnight at 42°C with 5′ radioactively labelled DNA probes complementary to R1 or R2 (see Appendix Table S1 for probe sequences). For probe synthesis, 10 pmol of each of R1 probe and R2 probe were labelled using T4 polynucleotide kinase (NEB) and 2 μl of [γ‐³²P] ATP (10 mCi/ml, 3,000 Ci/mmol, Hartmann Analytic). The following day, membranes were washed for 3 × 20 min with saline‐sodium citrate (SSC) buffer containing 0.1% SDS and imaged using a Typhoon FLA 9500.

Cell culture supernatant of DENV infected Vero cells under serum-free condition was collected 5 − 7 days post-infection and centrifuged at 15,000×g for 1 h, followed with 203,000×g for 4 h. The supernatant was subjected to immuno-affinity chromatography using anti-NS1 antibody (2G6) coupled to Sepharose 4B beads^{31 (link)}. Bound NS1 was eluted from beads with 20 mM diethylamine (DEA) pH 11.6 at a flow rate of 0.5 ml/min. The purity of eluted NS1 was assessed by SDS–polyacrylamide gel electrophoresis (PAGE). The protein concentration was determined by Qubit® protein assay (Thermo Fisher Scientific). Purified NS1 was stored in PBS (pH 7.4) and kept at − 80 °C until use.