Figure 6. BILF1 is physically associated with the MHC class molecule complex.

(A) 293 cells (10⁷) stably transduced with control (c) or BILF1 (b) retrovirus were metabolically labeled for 15 min and chased for 20 min. After lysis in NP-40 buffer and immunoprecipitation with mAb W6/32, the samples were dissociated by boiling in reducing sample buffer, and were re-precipitated with either 3F10 ( HA tag on BILF1) or HC10 (MHC class I heavy chain). Protein samples were separated by 10% acrylamide SDS/PAGE gel, dried and exposed to autoradiography. The arrowhead and bracket indicate the presence of 33 kD and 45–55 kD BILF1 bands, whilst the asterisks indicate probable ubiquitinated MHC class I species. (B) 293 cells (2×10⁶) stably transduced with control (c) or BILF1(b) retrovirus were treated with concanamycin A (50 nM) for 20 hr prior to lysing the cells with NP40 detergent buffer and immunoprecipitation with antibodies specific for MHC class I (W6/32), HA tagged BILF1 (12CA5), or TfR (H68.4). Cell lysates and immune complexes were separated by SDS/PAGE gel, and analyzed by western blotting using antibodies specific for MHC class I (HC10), HA tagged BILF1 (3F10), and TfR (H68.4). The first two samples on the gel are total cell lysates representing 5% of the input lysate for immunoprecipitations.