FIG. 3.

The ER is ruptured in SV40-infected cells. (A) Cleavage of the ER-resident membrane protein calnexin was monitored by immunoblotting with an antibody to the C terminus of calnexin (αCNX C-term). Confluent BS-C-1 cells were mock (M) or SV40 infected (I) and harvested as in Fig. 1A. (B) Immunoblots of the samples shown in panel A at 72 h postinfection (see lanes 17 and 18) were probed with an antibody to the N terminus of calnexin (αCNX N-term). (C) Diagram of the assay used in panel D showing the addition of trypsin to a cell with respect to the orientation of calnexin within the ER membrane. The larger schematic shows a more detailed topology map of calnexin in the ER membrane, depicting the location of the epitopes for the C- and N-terminal antibodies within the cytosolic and ER-luminal domains, respectively. N, nucleus; PM, plasma membrane; CNX, calnexin. Numbers indicate amino acid residues. (D) Mock (M) or SV40 infected (I) cells at 72 h were treated with trypsin for 15 min where indicated. The cell lysates were separated by SDS-PAGE and probed with the antibody to the C terminus of calnexin. The asterisk indicates a partially trypsinized form of calnexin. Number scale at left in panels A, B, and D indicates molecular mass in kilodaltons. P.I., postinfection.