Figure 6.

Binding of S100B to chondrocyte receptor for advanced glycation end products (RAGE). A, Human chondrocytes in monolayer culture were incubated for 2 hours at 4°C with biotin-labeled S100B, with or without soluble RAGE (sRAGE). Monolayers were washed to remove unbound protein, and cell lysates were prepared and then incubated with streptavidin–agarose beads to bind biotin–S100B and associated proteins (S100B pull-down). A sample of biotin–S100B that had not been incubated with cells was also incubated with the beads as an additional control. Proteins were eluted from the streptavidin beads in sodium dodecyl sulfate–polyacrylamide gel electrophoresis buffer and then were immunoblotted for RAGE with the monoclonal antibody. B, Primary chondrocytes transfected with a RAGE expression vector were also tested in the S100B pull-down assay and immunoblotted along with lysate samples from nontransfected cells (Cntl) and from transfected cells not subjected to the S100B pull-down assay. Arrows to the right of the blots indicate potential RAGE isoforms. Molecular weight markers are shown at the left.