Biovision plasma membrane protein extraction kit

In experiments of Figs. 2A, B, 3F, and 6J, the cells were subjected to Thermo Scientific Subcellular Protein Fractionation Kit (cat. No. 78840, Thermo) for separating cytosolic, total membrane, and nuclear fractions, following the manufacturer’s instructions. In experiments of Fig. 2E, the cells were subjected to BioVision Plasma Membrane Protein Extraction Kit (cat. No. K268-50/ab65400, BioVision) for separating plasma membrane and cellular organelles (non-plasma membrane from the bottom phase of step B4 of the manufacturer’s instructions), following the manufacturer’s instructions. In experiments of Fig. 6G, the cells were subjected to Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (cat. No. SM-005, Inventbiotech) for isolating plasma membranes and subjected to Minute™ Endosome Isolation and Cell Fractionation Kit (cat. No. ED-028, Inventbiotech) for isolating endosomes, following the manufacturer’s instructions.

Vero cells were either infected with DENV2 at a MOI of 2 or mock-infected and incubated at 37 °C, with 5% CO₂ for 10, 24, and 48 h. Plasma membrane proteins from the above cells were extracted separately, using a BioVision plasma membrane protein extraction kit (BioVision, USA). Approximately 50 µg of the plasma membrane fraction was resolved by 12% SDS-PAGE and transferred onto PVDF membranes, as described above. The membrane containing transferred protein was blocked with blocking buffer, at room temperature, for two hours, and probed with 1:1000 dilution of rabbit polyclonal anti-annexin II antibody, overnight, at 4 °C. The membrane was washed and incubated with 1:1000 dilution of rabbit anti-β-actin antibody (Cell Signaling Tech., Danvers, MA, USA), at room temperature, for two hours. The membrane was washed and incubated with a 1:9000 dilution of goat anti-rabbit HRP-conjugated secondary antibody (Abcam, UK), at room temperature, for two hours, washed, and then developed by ECL. All the washings were performed, three times, with TBS-T, for ten minutes each time. Images were acquired using a Syngene gel/chemi documentation system (Syngene, UK) and densitometry analysis of bands was undertaken using ImageJ software.