FIG. 6.

MLV NH slightly increases the level of degradation of MLV envelope protein, mainly through the proteasome pathway. (A) Two micrograms each of expression vectors for Env-HA and tTA were cotransfected into HEK293 cells with (left) or without (right) NH-YFPgpi (2 μg). Twenty-four hours later, these cells were treated with the proteasome inhibitor MG132 (20 μM), treated with the lysosomal degradation inhibitor NH₄Cl (20 mM), or not treated. After 10 h, cells were lysed, and the lysate was immunoprecipitated with anti-SU goat serum and blotted with anti-HA (top left and right) or anti-GFP (middle left) antibody. An aliquot of cell lysate that was not immunoprecipitated was blotted with anti-β-actin antibody (bottom left) as a control. Molecular mass markers (in kilodaltons) are shown at the left. IP, immunoprecipitation; IB, immunoblotting. (B) Transfected HEK293 cells corresponding to lanes 2 to 4 in panel A were cocultivated with U2OSLucCATGFP cells and assayed for luciferase activity. The x-axis label indicates inhibitor treatment. Error bars indicate the standard deviations of results with triplicate samples in a representative experiment of at least three independent transfections. RLU, relative light units.