FIG. 4.

Processing of the 8ab⁺ protein. vTF7-3-infected OST7-1 cells were transfected with the indicated constructs, in the presence (+) or absence (−) of tunicamycin (TM). The cells were labeled with ³⁵S-labeled amino acids from 5 to 6 h p.i., lysed, and processed for immunoprecipitation with specific antibodies followed by SDS-15% PAGE. (A) Cells were transfected with a construct encoding 8ab⁺-EGFP. The same construct was also in vitro transcribed and translated using the TNT coupled reticulocyte lysate system from Promega (ivt). Immunoprecipitations were performed with rabbit antiserum against the EGFP tag. (B) 8ab⁺-EGFP expressed in the absence of tunicamycin was treated with PNGase F or endo H after immunoprecipitation with rabbit serum against the EGFP tag. (C) Cells were transfected with a construct encoding 8ab⁺. The same construct was also in vitro transcribed and translated using the TNT coupled reticulocyte lysate system from Promega (ivt). Immunoprecipitations were performed with rabbit antiserum against 8ab⁺. (D) Cells were transfected with constructs expressing 8ab⁺ or 8ab⁺-EGFP, either full-length or after deletion of the predicted signal sequence (sig. seq.). The full-length constructs were also in vitro transcribed and translated using the TNT coupled reticulocyte lysate system from Promega (ivt). Immunoprecipitations were performed with rabbit antiserum against EGFP or 8ab⁺. (E) Cells were transfected with a construct encoding 8a carrying a wild-type EGFP tag (8a-EGFP) or with an EGFP tag containing an N-glycosylation site (8a-EGFP^glyc). The same constructs were also transcribed and translated in vitro using the TNT coupled reticulocyte lysate system from Promega (ivt). Immunoprecipitations were performed with rabbit antiserum against the EGFP tag. The positions and masses (in kDa) of the molecular mass protein markers are indicated. Only the relevant portions of the gels are shown.