Figure 2

Secretion of matrilin-3. A. Schematic diagram of MATN3 constructs. 1: wildtype MATN3, and 2: R116W mutant MATN3. The diagram below indicates the position of the point mutation in mouse MATN3 and its homology to human MATN3; a line indicates identical amino acid residues between mouse and human MATN3 while double dots indicate conserved changes of amino acid residues. B. Western blot analysis of recombinant matn-3. Cell lysate or conditioned medium of COS cells transfected with construct 1 or 2 was collected 48 hours after transfection, separated on a 10% SDS-PAGE under reducing conditions, blotted to a membrane, and incubated with antiserum against the V5 tag. Bound antibodies were detected with a peroxidase-coupled secondary antibody and a chemiluminescence detection kit. Cross-reaction to BSA in the medium samples containing serum is indicated. C. Time course of matrilin-3 secretion. Cos cells or MCT chondrocytes were incubated in the presence of 1% or 5% serum as indicated. Conditioned medium was collected at the indicated days after transfection, and analyzed on a 10% SDS-PAGE under reducing conditions. Western blot analysis was performed with antiserum against the V5 tag of the recombinant matrilin-3. In both COS cells and MCT chondrocytes incubated under different concentrations of serum, the quantity or the speed of the secretion of R116W MATN3 was diminished in comparison to the wildtype MATN3. D. Autoradiograph of matrilin-3 secretion in culture medium of MCT chondrocytes. MCT cells were transfected with wildtype (WT) or R116W mutant (MUT) matrilin-3 cDNA. Synthesized proteins were pulse-labelled with S-35 methionine for 1 hour and chased for 1 hour (1 h), 4 hours (4 h), 8 hours (8 h), and 24 hours (24 h). After each chase period, conditioned medium was collected for immunoprecipitation with an antibody against the V5 tag of the recombinant matrilin-3. Equal protein amount was loaded in each lane of the SDS-PAGE gel for autoradiogram analysis. E. Autoradiograph of recombinant matrilin-3 in the cell lysate and conditioned medium of matrilin-3 cDNA transfected Cos cells. Cos cells were transfected with wildtype (WT) or R116W mutant (MUT) matrilin-3 cDNA. Synthesized proteins were pulse-labelled with S-35 methionine for 1 hour and chased for 1 day (1 D), or 2 days (2 D). After each chase period, conditioned medium was collected and cells were lysed for immunoprecipitation with an antibody against the V5 tag of the recombinant matrilin-3. Equal protein amount was loaded in each lane of the SDS-PAGE gel for autoradiogram analysis.