Figure 2. Proteasomal involvement in VSG turnover.

Panel A: Western blot of cell lysates from trypanosomes exposed to 20µM MG-132 for the indicated times and probed for VSG221, ISG65 and ISG75, using polyclonal antibodies. “Con” indicates lysates from untreated cells, while MG132 lanes correspond to hours exposure to the compound. Lysates represent 1×10⁶ cell equivalents. Similar results were obtained with 10µM MG132, except that accumulation was less pronounced. The experiment was repeated three times with highly similar results. Equivalence of loading was monitored by reprobing of membranes with antibody to TbBiP. Panel B: Immunofluorescence analysis of trypanosomes either untreated (Con) or exposed to 20 µM MG123 for 1 and 4 hours. Following culturing cells were fixed, permeabilized and stained for VSG221 (red) and DNA (DAPI, blue). Note accumulation of VSG within the cell in the MG132-treated examples, which likely corresponds to aggresomes.