Figure 3.
Total protein synthesis and glycoprotein accumulation in tunicamycin-treated cell line overexpressing BiP. Wild-type (WT) and 35S-BiPS7 suspension cells were treated with the indicated concentrations of tunicamycin for 12 h and then were labeled for 3 h with [35S]Met and [35S]Cys. Incorporation of radiolabeled amino acids was measured by monitoring trichloroacetic acid (TCA)-precipitable radiolabeled proteins from [35S]Met and [35S]Cys-labeled cell lysates. A, Labeled glycoproteins were affinity purified using concanavalin-A-Sepharose resin and determined by liquid scintillation counting (Beckman Instruments, Fullerton, CA). Relative synthesis of glycoprotein was calculated by normalizing the TCA-precipitable activity (100%) in labeled cell lysates. Values are the mean ± sd from three replicates. B, Protein synthetic rates were calculated as the radioactivity incorporation per microgram of protein. Values are the mean ± sd from three replicates.