Pulse-chase experiments of ³⁵S-labeled rPLG. COS-1 cells were transiently cotransfected with either pTH3/WT (WT), pTH3/S572P (S572P), or pcDNA_neo (mock) and pSV–β-galactosidase plasmids. After pulse-labeling with L-[³⁵S] methionine for 30 minutes, the COS-1 cells then were chased for the times indicated (1 hour, 2 hours, 6 hours, 24 hours) in the presence of unlabeled methionine. In case of mock-transfected cells, culture medium and cell extract prepared 6 hours after pulse labeling were analyzed to examine the specificity of anti-PLG antibody. Both culture media (A, 1 mL) and cell lysates (B, 700 μL) were prepared, and immunoprecipitation samples were analyzed under nonreducing conditions as described above. β-Galactosidase present in the cell lysates was used as an internal control for both transfection and immunoprecipitation efficiency.