Figure 6.

Immunoprecipitation of endogenous protein in HepG2 cells further support the data that co-precipitation does not occur between the mature forms of OSTα and OSTβ. HepG2 cells were treated for 48 hrs with CDCA and then lysed with 1% Triton X-100 buffer as described in Methods. Cells extracts were subjected to immunoprecipitation using rabbit anti-OSTα (middle panel) or rabbit anti-OSTβ (right panel) and the immunoprecipitates (IP) and the fraction not bound to the Protein A/G beads (UB) were separated by SDS-PAGE. As a positive control, the cell lysate (Lys) was also subjected to SDS-PAGE (left panel). Western blot was then performed using the same polyclonal antibodies and a Native IgG kit from Pierce. In the case of HepG2 cells, only the mature form of OSTα is detected in the lysate. Co-immunoprecipitation of OSTα and OSTβ does not occur, although the proteins are clearly detectable in the unbound fractions. Lys = lysate; UB = unbound fraction; IP = immunoprecipitated, bead fraction