FIG. 5.

Proteasome inhibition increases the ubiquitination of complexes associated with late processing factors. (a) Complexes isolated before (−) and after (+) MG-132 treatment. 293EBNA cells were cotransfected with HA-ubiquitin and FLAG-NNP-1, FLAG-B23, FLAG-fibrillarin (FIB), or FLAG-nucleolin (NCL) as affinity baits. Complexes were separated by SDS-PAGE and silver stained. A star indicates the estimated location of the bait protein, based on its molecular mass as determined by the molecular mass standards (in kilodaltons) shown at the left side (see also panel c, which demonstrates the presence of the bait protein in each extract). (b and c) Western blots with anti-HA (ubiquitin tag) and anti-FLAG (bait tag) antibodies are shown. Mock, extract from cells transfected with the expression plasmid pcDNA3.1 without an inserted gene. (d) As an input control, the GAPDH content in each of the cell extracts is indicated at the bottom.