FIG. 3.

The display proteins are incorporated into retroviral particles. Display and capsid proteins were detected by Western blot analysis with 16% polyacrylamide gels or by ELISA. (A) HEK-293FT cells were transfected with the pHIT60 plasmid and pD-EGF (lane 1), pD-PrP111 (lane 2), pD-PrP209 (lane 3), or an unrelated control plasmid (lane 4). Cell extracts were analyzed with the anti-HA (αHA) antibody. (B) Supernatants of HEK-293FT cells transfected with pHIT60/pD-EGF (lane 1), pHIT60/pD-PrP111 (lane 2), or pHIT60/pD-PrP209 (lane 3) were concentrated by low-speed centrifugation. Pellets were resuspended in PBS. Western blotting was performed with the anti-HA antibody in the upper blot and with anti-p30 serum to detect the MLV capsid in the lower blot. Volumes loaded corresponded to the following amounts of cell culture supernatant: 0.7 ml (lane 1, upper blot), 0.35 ml (lane 2, upper blot), and 13.5 ml (lane 3, upper blot). Volumes loaded on the lower blot corresponded to 1 ml of cell culture supernatant each. (C) Supernatants of HEK-293FT cells transfected with pHIT60/pD-EGF (lane 1), pHIT60/pD-PrP111 (lanes 2, 3, and 5), pHIT60/pD-PrP209 (lane 4), or pHIT60/pD-PrP142 (lane 6) were detected with the anti-PrP antibody 6H4. For PNGase F digestion, particles were equilibrated in denaturing buffer and incubated in the presence (lane 3) or absence (lane 2) of PNGase F. Volumes loaded on the gels correspond to 0.7 ml (lane 1), 0.35 ml (lanes 2 and 3), 13.5 ml (lane 4), 0.8 ml (lane 5), and 1.2 ml (lane 6) of cell culture supernatants. (D) Analysis of PrP^D111 retroparticles (upper panel) and of EGF^D retroparticles (lower panel) by ELISA. Concentrated stocks of both particle types were applied to ELISA plates at the indicated dilutions. Virally expressed antigens were detected by use of the anti-PrP monoclonal antibody 6H4, polyclonal anti-p30 antiserum, or mouse preimmune serum. Data represent results from one of two experiments with similar results.