6×His-tagged protein precipitation.

BSR-T7 cells (2.5 × 10⁶) were seeded into 100-mm culture dishes and cotransfected with 9 μg of an equal mixture of three plasmids coding for hemagglutinin-tagged ubiquitin (HA-Ub) (39), MeV P, and either a Flag-tagged or a Flag- and 6×His-tagged MeV L protein. Seven hours later, 2 μM 17-DMAG and 5 μM MG132 were added or not added. At 24 h posttransfection, the cells were lysed in 1 ml of DNPI-10 denaturing buffer (8 M urea, 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0), incubated for 20 min on ice, sonicated twice for 15 s each time, and centrifuged for 15 min at 15,000 × g and 4°C. The supernatant was incubated on Ni-nitrilotriacetic acid (NTA) agarose beads (Protino Ni-NTA Agarose; Macherey-Nagel) for 6 h. The beads were washed and eluted as recommended by the manufacturer. Loading buffer was added prior to SDS-PAGE and Western blot analysis.