The secretion of mutant COMP is delayed but not abolished. (a) The secretion of wild-type (Wt) or mutant (Mu) COMP in monolayer chondrocytes transduced with 200 MOI was analyzed after c-myc specific immunoprecipitation in pulse-chase experiments. Ly, cell lysates; Su, supernatants. The hours of chase are indicated below the gels. Molecular weight standards are shown on the left, the position of reduced recombinant COMP on the right. Arrows indicate origin and front of the separating gel. (b) Cell lysates of chondrocytes treated as above and chased for 30 minutes were subjected to gel electrophoresis under nonreducing conditions after c-myc specific immunoprecipitation. The position of recombinant COMP multimers is indicated on the right side of the gel. Ψ4-mer indicates a pseudotetramer (19). (c) A pulse chase experiment of one-week-old alginate cultures transduced with 200 MOI followed by COMP-specific immunoprecipitation shows the proportion of COMP found in the cell lysate versus total COMP. Closed symbols represent mutant COMP, open symbols wild-type COMP, shaded symbols endogenous COMP from untransduced chondrocytes. (d and e) Chondrocytes were transduced with 20 (d) or 200 (e) MOI, cultured in alginate for five weeks, and pulse-labeled. The COMP-specific precipitated activity in supernatant (Su), citrate soluble alginate fraction (Ci), and cell lysate (Ly) is indicated. Black bars represent cultures expressing mutant COMP (Mu), white bars represent wild-type COMP (Wt), and gray bars represent controls (C). The insets show radiographs of the gels.