Fig. 8. Stimulated secretion of lysosomally matured CB-EGFP from FRTL-5 cells. Lysates of non-transfected (A, lane 1) or CB-EGFP-expressing FRTL-5 cells (A, lane 2) were normalized to provide equal amounts of protein and separated on 12.5% SDS gels for subsequent blotting and immunolabeling with antibodies against the propeptide of rat cathepsin B. Autoradiography of 12.5% SDS gels of secretion media from CB-EGFP-expressing, G418 selected FRTL-5 cells after 1 hour of pulse radiolabeling (B, lanes 3 and 4) and chasing for the indicated time intervals in media without TSH (B, 5H, lanes 5-10) or with 50 µU/ml TSH (B, 5H + TSH, lanes 11-16). Immunoprecipitation was with antibodies against rat cathepsin B (CB, odd numbered lanes in B) or against the propeptide of rat cathepsin B (PP, even numbered lanes in B). Molecular mass markers are given in the margins. The positions of the proform (proCB-EGFP) and the mature chimeric protein (CB-EGFP), as well as of procathepsin B (pro) and single chain cathepsin B (SC) are indicated in the margin between A and B. In C, the proteolytic activity of CB-EGFP secreted from continuously TSH-stimulated FRTL-5 cells after immunoprecipitation with anti-GFP antibodies is given as mean±standard deviation. Note that antibodies against the propeptide of cathepsin B failed to immunoprecipitate CB-EGFP from the secretion media (B) and that secreted CB-EGFP was proteolytically active (C).