Figure 8.

Effect of reduction and heat denaturation on the ability of lubricin to inhibit Prg4^–/– synoviocyte growth. Portions of tissue culture plates (to the right of the dashed lines) were incubated overnight at 37–C with untreated (A), DTT-reduced (B), or heat-denatured (C) lubricin media. After washing off the media with several changes of PBS, we added Prg4^–/– synoviocytes to the culture dishes and allowed them to proliferate in growth media for 7 days. Note that cells were able to proliferate on the portions of the plates that contained reduced or heat-denatured lubricin media but not on untreated lubricin media (magnification, ×100). (D–F) Immunodetection of lubricin adhesion to tissue culture plastic. Upper wells were incubated overnight at 37–C with untreated (D), DTT-reduced (E), or heat-denatured (F) control media. Lower wells were incubated overnight at 37–C with untreated (D), DTT-reduced (E), or heat denatured (F) lubricin media. Wells were washed several times with PBS, and we determined lubricin adhesion to the plastic using an anti-lubricin antibody and an HRP-conjugated secondary antibody. Note that lubricin was detected in all 3 lubricin media wells and not in any of the control media wells.