Figure 3.

Immunoprecipiation of GERp95 from cultured cells. (A) NRK52E (N) and BHK-21 (B) cells were biosynthetically labeled with ³⁵S cysteine/methionine for 6 h before lysis and immunoprecipitation with LCH-7 or a mAb to CD8 ((−) Ab). Samples were subjected to SDS-PAGE on 8% gels followed by fluorography. A 95-kDa protein, GERp95 (arrowhead), is immunoprecipitated from both cell types with the LCH-7 antibody (lanes 1 and 2) but not the negative control Ab (lanes 3 and 4). ¹⁴C-labeled protein standards (kDa) are shown lane M. (B) NRK52E cells were biosynthetically labeled with ³⁵S as above in the presence and absence of tunicamycin (3 μg/ml) before lysis and immunoprecipitation with LCH-7. Cells were infected with VSV and radiolabeled using the same conditions, and the VSV G protein was immunoprecipitated and subjected to SDS-PAGE and fluorography. The mobility of VSV G is increased in the presence of tunicamycin attributable to inhibition of N-linked glycosylation.