Figure 10.

HCT-116 cells were washed twice with PBS and incubated in the presence of indicated concentrations of compounds under media lacking methionine and cysteine for 30 min. ³⁵S-labeled methionine and cysteine were then introduced to the media, and cells incubated for a further 90 min. Cells were harvested by scraping into ice cold PBS and analyzed by SDS-PAGE and autoradiography. (A) The collected cells were subsequently homogenized and analyzed by SDS-PAGE and autoradiography. (B) As for (A) but the samples are the cytosolic contents of harvested cells following partial permeabilization with 0.15% digitonin. (C) As for (A) but the samples are from TCA-precipitated culture medium from the same experiment. Ipom-F denotes ipomoeassin F. AprA denotes control samples treated with apratoxin A to block protein translocation into the ER. CHX denotes samples treated with cycloheximide and chloramphenicol to inhibit total cellular protein synthesis. Cotransin CT8 is a substrate-selective inhibitor of protein translocation into the ER.