Figure 2.

Impact of MVB adaptors and ESCRT-I on MLV VLP and virion release. (A) HuH-7 cells were treated with siRNA duplexes targeting Tsg101, Alix, Nedd4-1, or control siRNA for 48 h and were retransfected with the MLV constructs. In cases where single siRNA duplexes were used, the target gene is designated with a star. After transient expression for 24 h, lysates were subjected to Tsg101-, Alix-, and Nedd4-specific WB to monitor the efficiency of depletion/cross-depletion. Non-specific bands stained by the antisera served as controls for gel loading (Load); (B) The siRNA-treated cells were transfected with MLV.Gag^YFP. Lysates (Cell) and VLPs released into the supernatants (SPN) were analyzed by anti-GFP WB. Uniformity of sample loading was probed by anti-ß-Actin WB. VLPs values were determined by densitometry and demonstrated in percent amount relative to control cells; (C) siRNA-treated cells were retransfected with the retroviral MLV.WT^FLAG/HA construct encoding FLAG-tagged Gag and HA-tagged Env proteins. Cell extracts and supernatants were analyzed by FLAG- and HA-specific WB. For a loading control, lysates were reacted with anti-ß-Actin antibodies. For quantification of extracellular virions, Env- and Gag-specific signals were measured by densitometry, summed, and demonstrated in percent amount relative to control cells.