Fig. 4. Ubiquitination of clusterin. (A) 48 hours after transfection with GFP--clu or GFP-SP-clu, COS-7 cells were fixed, permeabilized and incubated with antibody against ubiquitin, coupled with secondary rhodamine-conjugated antibody. Diffused and aggregated GFP fluorescence closely co-localized with ubiquitin and this co-localization was systematically observed. Bar, 10 µm. (B) COS-7 cells were co-transfected with GFP-SP-clu, pCMV-ß-gal and expression plasmids encoding ubiquitin, wild-type (Ubi) or mutated (K48R-Ubi), or the empty vector (Control). 36 hours after transfection, extracts from cells treated (+) or not (-) with MG-132 (10 µM for 6 hours) were prepared by 100,000 g ultracentrifugation. Supernatants and pellets were analysed by western blotting with a clusterin antibody after normalization of cell extracts with ß-galactosidase. In addition to the expected molecular weight of GFP-SP-clu between 70 kDa and 80 kDa, high molecular weight forms (hmw) appeared after MG-132 treatment. These forms were no longer visible when using the polyubiquitin chain terminator K48R mutant ubiquitin and probably correspond to ubiquitinated clusterin.