Figure 11.

Rab24 is not conjugated to ubiquitin. HEK293 cells were transfected with vectors encoding His₆-tagged Rab24wt or Rab24(D123). (A) The expressed proteins were extracted in high-detergent buffer, purified on Ni⁺⁺ spin columns, and subjected to SDS-PAGE and immunoblot analysis using a monoclonal antibody against the His₆ epitope. Cells were incubated with (+) or without (-) 20 μM lactacystin for 4 h prior to harvest. (B) Separate aliquots of the proteins collected from the Ni⁺⁺ columns (cells treated with lactacystin in panel A) were immunoblotted with a monoclonal antibody against ubiquitin. To confirm the reactivity of the antibody, it was also applied to a blot containing an aliquot of total protein from untransfected cells treated with lactacystin for 4 h (cell lysate). The exposure time was the same for all blots.