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. 1999 Mar 2;96(5):2104–2109. doi: 10.1073/pnas.96.5.2104

Figure 5.

Figure 5

In vitro binding assay of PAHX with immunophilin. (A) Human PAHX specifically binds FKBP52 in vitro. In vitro binding assays were carried out by incubation of 500 pmol GST-FKBP52 resin (lanes 1–3), 500 pmol GST-FKBP12 resin (lanes 4–6), or 500 pmol GST resin as control (lanes 7–9) in the presence of increasing quantities of PAHX. The molar ratio of PAHX added to different resins is indicated under each lane. After processing the resin as described in Material and Methods, the presence of PAHX retained on the GSH beads was analyzed by Western blotting by using a polyclonal antibody directed against PAHX. Recombinant purified PAHX is loaded as control in lane 10. (B) The PAHX/immunophilin complex formation is not modulated by FK506 in vitro. (Upper) GSH beads loaded with 500 pmol of GST-FKBP52 were treated with increasing quantities of FK506 as indicated under each lane, before incubation with 500 pmol of PAHX. Bound proteins were subjected to Western blotting analysis by using a polyclonal antibody directed against PAHX. Lane 1, no PAHX was added to the resin. (Lower) GSH beads loaded with 500 pmol of GST-FKBP52 (lanes 1–3) or 500 pmol of GST-FKBP12 (lanes 4–6) were treated or not with 10 nmol of FK506 before incubation with 500 pmol of PAHX, as indicated in Materials and Methods. Bound proteins were subjected to Western blotting analysis by using a polyclonal antibody directed against PAHX. Lanes 1 and 4, no PAHX was added to the resin. Recombinant purified PAHX was loaded as control in lane 7. Migration of molecular size markers (New England Biolabs) is indicated on the left.