Figure 5.

Immunoprecipitation of IL-17R ECD with IL-17, IL-17B, and IL-17C. His-tagged IL-17 receptor ECD was expressed in 293 cells and metabolically labeled with ³⁵S as described in Materials and Methods. The supernatant was collected. To examine the expression of the IL-17R ECD, Ni-NTA beads were used to affinity-precipitate the His-tagged IL-17R ECD in the supernatant (lane 1). (A) IL-17, IL-17B.Fc, IL-17C.Fc, or the control Fc fusion protein was incubated with the supernatant, and protein A-agarose beads were added to precipitate the Fc fusion proteins. For IL-17 immunoprecipitation reaction, anti-IL-17 antibodies were included. (B) For the competitive binding experiment, immunoprecipitation of IL-17R ECD by IL-17 was performed in the presence of a 5-fold excess of IL-17B.His and control His-tagged proteins. Precipitates in both A and B were analyzed by electrophoresis on NuPAGE (4–12% Bis-Tris) gels. Molecular mass markers are indicated at the left of A and B.