Fig. 4.

Subcellular fractionation of ubiquitinated Jun. (A) Nuclear (N) and cytoplasmic (C) fractions were prepared from cells that expressed Jun and ubiquitin tagged with the Xpress and hemagglutinin (HA) epitope tags, respectively. Aliquots of the fractions were analyzed by Western blotting with anti-Xpress antibody to detect total Jun (Left). The fractions were immunoprecipitated by using anti-Xpress antibody, and the immunoprecipitates were analyzed by Western blotting by using anti-HA antibody to detect ubiquitinated Jun (Right). (B) The cytoplasmic extract was fractionated by density gradient sedimentation. Total Jun (Top) and ubiquitinated Jun (Middle) in each fraction was analyzed as described in A. (Bottom) Lysosomal β-hexosaminidase activity in each fraction is plotted as a percentage of the total activity applied to the gradient.