FIG 3.

Treatment of MG132 diminishes the NE modulation and vesicle formation abilities of EBV BFRF1. (A) The steady-state expression level of EBV BFRF1 was not enhanced by MG132 treatment. HeLa cells were transfected individually or cotransfected with HA-BFRFl-, Myc-Ub-, or Myc-Ub48R-expressing plasmids. At 6 h posttransfection, the cells were treated with DMSO solvent or 10 μM MG132 for 18 h. The cell lysates were harvested and immunoprecipitated (IP) using antibody against HA or GST as a negative control. The immunocomplexes were detected by antibodies against Myc and HA. (B) Confocal images of HeLa cells transfected with HA-BFRF1, together with vector control or Myc-Ub, and treated with DMSO solvent (left) or 10 μM MG132 (right) for 18 h. HA-BFRF1 and emerin were visualized by indirect immunofluorescence. The boxed regions are enlarged below. (C) Overexpressed HA-BFRF1 protein in HeLa cells was partially purified by immunoprecipitation with HA antibody, separated by SDS-PAGE, and subjected to mass spectrometric analysis. IgH and IgL indicate the immunoglobulin heavy and light chains. M, molecular mass marker. (D) The recovered BFRF1 fragments were identified by Mascot software against the NCBI virus protein database. The coverage rate of the BFRF1 fragment was 63% (red letters). Shown is ubiquitination of Lys-120 (K120) in the recovered BFRF1 103 to 120 fragment.