Figure 9.

A reagent of about 5 kDa cannot pass the ER membrane. HeLa cells (2 × 10⁶) were permeabilized with 0.04% digitonin or lysed in 1% Triton X-100 as indicated. Modification was carried out for 30 min at 4°C with 1.5 mM mPEG, an SH-reagent of ∼5 kDa. Where indicated, DTT was present during the reaction. Samples corresponding to 5 × 10⁴ untreated (T) or mPEG-modified cells were analyzed by SDS-PAGE on a 7.5-17.5% urea gel, followed by immunoblotting with antibodies to GRP94 or Sec61β. xSec 61β and xGRP94 indicate the positions of the modified proteins.