FIG 4.

L and P proteins associate into soluble complexes in the presence of HSP90 activity. (A) Schematic view of the experiment. BSR-T7 cells were transfected with plasmids, and 6 h later, cells were treated with 2 μM 17-DMAG and/or 5 μM MG132 or not treated. Cells were lysed in urea-free lysis buffer at 28 h posttransfection, and soluble proteins were separated by centrifugation at 15,000 × g for 15 min (soluble fraction). The pellet was then washed twice in urea-free lysis buffer and incubated in 8 M urea-containing buffer. Denatured protein aggregates were finally separated after 15 min of centrifugation at 15,000 × g (insoluble fraction). (B, C) Western blot analysis of soluble and insoluble fractions from BSR-T7 cells cotransfected with L and a control (ctrl.) plasmid (left) or a P plasmid (right). Data for MeV are in panel B, and those for VSV are in panel C.