Western blot analysis: GP expression in infected cells and incorporation in the rHPIV1 particles.

Vero cells were infected at an MOI of 3 TCID₅₀ with the four constructs, including wt HPIV1 and HPIV1-C^Δ170 empty vector as controls, and incubated at 32°C for 48 h. Cell lysates were prepared by direct lysis of the monolayer with 100 μl of 1× lithium dodecyl sulfate (LDS) sample buffer, and 22.5 μl of lysate was reduced, denatured, and electrophoresed on a 4 to 12% bis-Tris SDS gel (Life Technologies). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and analyzed by Western blotting.

To analyze EBOV GP incorporation in the HPIV1 vector virions, LLC-MK2 cells were infected at an MOI of 0.1 TCID₅₀ per cell with viruses expressing EBOV GP, wt HPIV1, and rHPIV1-C^Δ170 empty vector and incubated at 32°C for 6 days. The medium supernatant was clarified, and virus was purified by discontinuous sucrose gradient centrifugation. The protein content of each purified virus was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). One microgram of protein of each sucrose-purified virus preparation was lysed in radioimmunoprecipitation assay (RIPA) buffer and subjected to SDS-PAGE and Western blot analysis.