FIGURE 6. ER oxidoreductases interact simultaneously and covalently with Tg-G2320R.

A, transiently transfected COS-7 cells expressing Tg-G2320R were pulse-labeled and chased for 0 and 6 h. Cells were lysed and first immunoprecipitated with an antibody against Tg, ERp72, or PDI as indicated; the recovered Tg was analyzed in lanes 1, 5, and 9, respectively. The supernates of these IPs were then used as the starting material for a fresh second round of immunoprecipitation with antibodies against Tg, ERp72, or PDI. First and second round immunoprecipitates were analyzed by reducing SDS-4% PAGE and phosphorimaging. Only the labeled Tg band recovered is shown. B, transiently transfected COS-7 cells were pulse-labeled and chased for the times shown before cell lysis on ice for 1 h in the presence of apyrase (see “Experimental Procedures”). After a brief centrifugation to remove cell debris, the lysate was split into two equal portions; one was left untreated (left panels), and SDS was added to the other to a final concentration of 2% (right panels). After a 30-min incubation, all samples were diluted 10-fold in buffer lacking SDS (but containing 1% Triton X-100) prior to immunoprecipitation with the antibodies shown.