Figure 4.

Effect of anti-CD47/IAP antibody on chondrocyte responses to mechanical stimulation. (a) Cell lysates from normal human articular chondrocytes at rest or mechanically stimulated at 0.33 Hz for 1 minute in the presence of either non-immune mouse immunoglobulin (Ig) or the function-blocking CD47/IAP antibody Bric 126 were immunoprecipitated with anti-phosphotyrosine and immunoblotted with mouse monoclonal antibody anti-phosphotyrosine-horseradish peroxidase, PY-20, at 1:1,000. Identical amounts of whole-cell lysates were used for immunoprecipitation of phosphotyrosinated proteins from stimulated and unstimulated cells. Tyrosine phosphorylation of a 125-kDa protein is increased after 1 minute of mechanical stimulation but this response is diminished by the presence of the anti-CD47/IAP antibody Bric 126. (b) Following 0.33-Hz mechanical stimulation for 20 minutes in the presence of control non-immune mouse Ig or anti-CD47/IAP antibody Bric 126, relative levels of aggrecan mRNA were assessed in cells immediately and 1 and 3 hours after incubation at 37°C. Consistent with previous results [7], mechanical stimulation of normal articular chondrocytes in the presence of non-immune mouse Ig results in an increase in relative levels of aggrecan mRNA. This response is inhibited by the presence of the anti-CD47/IAP antibody Bric 126. *p < 0.05, 1 hour after mechanical stimulation versus immediate post stimulation. **p < 0.05, 1 hour after stimulation in the presence of Bric 126 versus 1 hour after stimulation in the presence of non-immune mouse Ig. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IAP, integrin-associated protein; MS, mechanical stimulation.