Figure 1.

Sample preparation and the presence of PV proteins in secreted vesicles. (a) Schematic of collection and purification of microvesicles and exosomes. Annexin-V-coated magnetic beads were used to purify microvesicles that include phosphatidylserine (PS) in their outer membrane. (b) Cell viability at the time of vesicle collection, 8 hpi, showing minimal cell death. (c) Mass spectrometric analysis of poliovirus protein abundance in isolated infectious microvesicles collected at 8 hpi. Protein abundance was normalized to the GAPDH level of PV-infected cells, multiplied by 1000 and presented as log₁₀ (Normalized Abundance). (d) Viral structural proteins (VP0, VP1, VP2, VP3) and non-structural proteins (2C, 2BC 3D, 3CD, 3A, 3AB) were identified in PS-containing infectious microvesicles (ImVs), detected via western blot using polyclonal antibodies against 2C, 3A, 3D, and/or GAPDH, from samples taken at 5 hpi (anti-3A) or 8 hpi. For each antibody used, the ImV and mock-infected microvesicles (MmV) lanes were run on the same gel, and the same vertical position on the gel is shown for both lanes. Full western blots from which the lanes were taken are all shown in Supplementary Fig. S2: VP proteins, Suppl Fig. S2a; 2C protein, Suppl Fig. S2b; 3D protein, Suppl Fig. S2c; 3A protein, Suppl. Figure S2d.