Analysis of calreticulin biosynthesis in vitro. Cell-free protein synthesis was performed in the rabbit reticulocyte lysate system in the presence of 35S-methionine. Either immunoprecipitates (A and B) or total translation products (C) were analyzed by 7.5% SDS-PAGE and fluorography. The locations of molecular weight markers are indicated. (A) Reactions contained either no RNA (−) or poly-A–enriched RNA from HL-60 cells (+) and either no membranes (−) or dog pancreatic microsomes (+). The products of protein synthesis were immunoprecipitated with either nonimmune serum (NIS; from the rabbit subsequently immunized with calreticulin), anticalreticulin (CRT), or antimyeloperoxidase (MPO). (B) Reactions were as in (A) with (+) or without (−) HL-60 cell RNA and dog pancreatic microsomes. HL-60 cell calreticulin biosynthetically labeled as in Fig 2 was run (third lane from left) for comparison with the cell-free products. For all samples products of translation were immunoprecipitated with anticalreticulin.
(C) Reactions contained no RNA (−) or synthetic mRNA generated from plasmid templates of the cDNA for either calreticulin (CRT) or myeloperoxidase (MPO).
