Figure 1.

Surface reexpression assay. (A) Schematic of the surface reexpression assay. Proteins expressed on the cell surface are removed by treating cells with extracellular proteases. Protease-treated cells are then placed into culture at 37°C to allow ongoing protein transport to reestablish surface expression, which is evaluated biochemically by biotin labeling. It should be appreciated that proteins might also become accessible to labeling if there is cell death during culture. To distinguish legitimate surface reexpression from artefactual internal labeling, protease-treated cells are cultured in parallel with the protein transport inhibitor BFA, which blocks surface reexpression by viable cells, but not internal labeling of cytosolic proteins in dying cells. (B) An immature thymocyte cell line was either mock treated (HBSS, 15 min at 37°C) or protease treated (2 mg/ml pronase/HBSS for 15 min at 37°C), following which cells were cultured in complete medium at 4°C. In addition, proteolyzed cells were cultured at 37°C in complete medium alone or supplemented with 1 μg/ml BFA following which the cells were surface labeled with biotin, lysed in digitonin lysis buffer, and immunoprecipitated with anti-CD3ɛ mAb (145–2C11). The immune complexes were resolved on SDS/PAGE gels, blotted to Immobilon poly(vinylidene difluoride) membranes and visualized with HRP-Av and chemiluminescent detection. The same membranes were then washed in PBS-Tween, reblocked in 5% milk/PBS, and immunoblotted with the immunoprecipitating Ab to determine the effect of treatment on total protein levels. Bound Ab was visualized with ¹²⁵I protein A and autoradiography. The immature thymocyte cell line used in these experiments was the VL3–3M2 thymic lymphoma. Similar results were obtained with primary thymocytes from tissue explants. (C) The surface reexpression assay was performed as above except that a parallel culture supplemented with the protein synthesis inhibitor, CHX, was added and the resultant detergent extracts were immunoprecipitated with rabbit anti-calnexin Ab reactive with calnexin’s lumenal domain (anti-cal-N). (D) The surface reexpression assay was performed as above using rabbit anti-calnexin Ab reactive with calnexin’s C-terminal 12 aa (anti-cal-C, Left) and calnexin’s lumenal domain (anti-cal N, Right). It should be noted that the surface-labeled species migrating beneath calnexin (Right) may represent breakdown products possibly generated during turnover of surface calnexin. Consistent with this explanation, breakdown products are not seen among newly reexpressed calnexin molecules (lane 9) suggesting that they result from degradation of calnexin molecules after they have reached the cell surface.