Synthesis and secretion of FVIII WT and^ARG531^HIS expressed in COS-1 cells. FVIII WT and ^ARG531^HIS plasmids were transfected into COS-1 monkey cells. At 64 hours posttransfection, cells were pulse-labeled with [³⁵S]-methionine for 30 minutes and cell extracts were harvested. Duplicate labeled cells were chased for 4 hours in medium containing excess unlabeled methionine and then cell extracts and conditioned medium were harvested. Equal proportionate volumes of cell extract (lanes 1 through 6) and conditioned medium (lanes 7 through 9) were immunoprecipitated with anti–FVIII-specific antibody and equal aliquots were analyzed by SDS-PAGE. Mock indicates cells that did not receive plasmid DNA. Cell extract pulse (P) and chase (C). The migration of FVIII from the cell extracts is indicated at the right by an arrow. FVIII from the conditioned medium is indicated at the far right as single-chain (SC), heavy-chain (HC), and light-chain (LC) forms. Molecular weight markers are shown on the left.