Intracellular retention of L353R and G400D mutant fibrinogens.
COS-1 cells were incubated with l-[35S] methionine and l-[35S] cysteine for 3 hours and then chase incubated with 10 mmol/L l-methionine andl-cysteine for various periods of time up to 3 hours. At the specified chase periods (0, 30, 90, and 180 minutes), radioactive fibrinogen was immunoprecipitated from cell lysates and from the corresponding culture media, as described in “Materials and Methods.” Intracellular (A) and secreted (B) immunoprecipitable proteins were separated by 4% SDS-PAGE under nonreducing conditions. The arrowheads indicate the 340-kd fibrinogen complex. Intracellular and secreted fibrinogen at the end of the pulse period from control HepG2 cells was also analyzed. In both panels, pUC18 lane contains immunoprecipitable proteins at the end of the pulse period from COS-1 cells transfected with the unrelated pUC18 plasmid.
