Figure 7.

Colocalization of Parkin inclusions with other components of the ubiquitin-proteasome pathway. (A–R) COS-7 cells were transfected with the FLAG-Parkin expression construct and cultured in the presence or absence of MG132 for 16 h. Cells were probed with the following: A–F, anti-FLAG and affinity-purified rabbit anti-UbcH7 peptide (1:100) antibodies; ubiquitin (1:50) antibodies; G–L, anti-FLAG and anti-20S (1:100) antibodies; or M–R, anti-FLAG and anti-ubiquitin (1:50) antibodies. Staining was as follows: left, FLAG, to identify Parkin (red); middle, UbcH7 (B and E), 20S (H and K), or ubiquitin (N and Q) (green); and right, overlay of each set with the addition of 4,6-diamidino-2-phenylindole staining (blue) to identify the nucleus. Regions of colocalization within cells stain yellow. Arrows indicate colocalization within an inclusion. Bars, 20 μm. (S) COS-7 cells were transfected with the Myc-Parkin expression construct alone or cotransfected with the FLAG-tagged ubiquitin construct as indicated and cultured in the absence of MG132. Cells were analyzed by immunofluorescence by using anti-Parkin antibodies as described for Figure 1 and assessed as described in Figure 3. Error bars indicate the SE from the mean. The asterisk (^*) indicates a significant difference between the number of Myc-Parkin/FLAG-ubiquitin construct-transfected cells containing inclusions versus the number found in Myc-Parkin alone transfected cells; p < 0.01. Costaining with anti-FLAG antibody revealed that exogenous ubiquitin had an identical staining pattern to the endogenous protein (our unpublished data).