Proteasome Subunit Interactions Probed by Pull-Down Assays

The 3xFLAG-tagged α4, GST-fused proteasome α5-subunit (GST-α5), non-tagged or His₆-tagged forms of proteasome α1, α2, α3, α6, and α7 subunits, and scPAC3/4 were used in the pull-down assays. For immobilization, 20 μg of His₆-tagged scPAC3/4 or GST-α5 was applied to Ni²⁺-charged Chelating Sepharose or Glutathione Sepharose 4B (GE Healthcare) resins, respectively. The His₆-scPAC3/4-immobilized resins were incubated with 50 μg of α1–α7 subunits for 2 h at 4 °C in an incubation buffer (20 mM Tris-HCl (pH 8.0) and 150 mM NaCl). For α5, the GST-α5-immobilized resins were incubated with 50 μg of α1–α4, α6, and α7 in the presence and absence of 50 μg of scPAC3/4 as described above. Since α7 makes a stable complex with α6 [19 (link),20 (link)], the pull-down experiments containing α7 were performed separately. The resins were washed four times with the incubation buffer, which contains 60 mM imidazole in the His₆-tag pull-down assays. Proteins bound to the Chelating or Glutathione Sepharose resins were eluted using 20 mM Tris-HCl (pH 8.0)/500 mM imidazole or 50 mM Tris-HCl (pH 8.0)/10 mM reduced glutathione, respectively, and analyzed by SDS-PAGE, stained with CBB.