Figure 8.
Multiprotein complex remains upon treatment with cycloheximide to inhibit the translation of new proteins. Ag8.653 cells (8 × 106) were left untreated or treated with 50 μg/ml cycloheximide for 2 h. The postnuclear fraction was prepared from cells, cross-linked with DSP, and lysed in NP-40 lysing buffer. The two cross-linked ER lysates were divided into five aliquots, each of which were directly applied to 5–15% gradient gels before transferring to nitrocellulose membranes for blotting. Untreated Ag8.653 cells (8 × 105) were lysed without prior cross-linking to identify the mobility of the free pool of the various proteins. The nitrocellulose membranes were reacted with the indicated polyclonal antisera. The mobility of the free pool of each protein is indicated with an arrow.