Fig. 7. Lysosomal CB-EGFP and its secretion from transfected cells. Lysates of lysosomal fractions of non-transfected (lanes 3, 5, 7, 10, 12, 14) or CB-EGFP-expressing CHO, FRT or FRTL-5 cells after selection with G418 (lanes 4, 6, 8, 11, 13, 15) were normalized to contain equal amounts of protein and separated on 12.5% SDS gels. After blotting, proteins were immunolabeled with antibodies against rat cathepsin B (A) or GFP (B). Recombinant human procathepsin B (lane 1), bovine spleen cathepsin B (lane 2) and EGFP (lane 9) were used as standards. C shows an autoradiograph of SDS-PAGE-separated anti-GFP immunoprecipitates from culture media collected after the indicated time intervals from radiolabeled non-transfected (lane 18) or CB-EGFP-expressing FRT cells (lanes 16 and 17). Molecular mass markers are given in the left margin. The positions of the intact chimeric protein (CB-EGFP) and its degradation fragment (F1) as well as those of procathepsin B (pro), single chain (SC) and heavy chain of two-chain cathepsin B (HC) are indicated in the right margins. The proteolytic activity of cathepsin B within conditioned media of transfected (+) or non-transfected CHO, FRT or FRTL-5 cells was determined at pH 6.0 by using a colorimetric assay (D). Cathepsin B activities in D are given as mean±standard deviation; levels of significance are indicated as ^** for P<0.01, n=3.

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