VLP-Antibody Binding Interaction Assay

Antibodies (gal-3, ALIX, LAMP-1, Rab11, and Rab14) were incubated with 5 µg of GII.4 VLPs for 1 h at room temperature. An antibody pull-down was performed by adding 25 µl of PBS-washed protein A/G magnetic beads (#88802, ThermoFisher Scientific) to the mixture and incubating the mixture for 30 min at room temperature. The beads were collected by placing tubes on a magnetic stand and the unbound proteins present in the supernatant were collected for analysis. The pelleted beads were washed with PBS (5 times) and the bound proteins were analyzed after adding 100 µl SDS-PAGE buffer and heating the beads at 95 ^oC for 5 min (pull-down fraction). The supernatant and pull-down fractions were assessed for the presence of VLPs by SDS-PAGE, followed by Western blot analysis for the capsid VP1 protein (Fig. S6a).