Left: Immunofluorescent analysis of HeLa cells stained with rabbit antibody to ubiquitin (red, 1:1,000) by Immunocytochemistry. Cells were co-stained with chicken antibody to vimentin (C-1409-50, green, 1:10,000). Blue: DAPI nuclear stain. [A] Control HeLa cells maintained in normal medium; [B] HeLa cells treated with 10 uM of the proteasome inhibitor lactacystin (Lc) for 24 hours. Proteasomal inhibition leads to formation of strongly ubiquitin positive cytoplasmic inclusions [B]. Note the diffuse cytoplasmic ubiquitin staining in control cells [A] and well-defined ubiquitin positive inclusions in the Lc treated cells [B]. Right: Western blot analysis of HEK293 cell lysates using rabbit antibody to ubiquitin (green, 1:5,000).  protein standard,  cells maintained in normal medium,  cells treated with proteasome inhibitor lactacystin (Lc) at 10 um for 16 hours. The blot was simultaneously probed with a mouse antibody to beta-tubulin as loading control (red, lanes 2 and 3). The smear detected above the 200 kDa standard represents accumulations of ubiquitinated proteins in the Lc-treated cells. The prominent band at ~8 kDa corresponds to monoubiquitin.