Fig. 3

Parkin interacts with and ubiquitinates cav-1. a Parkin KO MEF cells were transfected with plasmids for flag-parkin and cav-1-EGFP for 48 h, and then the lysates were immunoprecipitated with anti-flag and anti-EGFP antibodies, respectively, followed by Western blotting. b WT MEF cells cultured on poly-D-lysine-coated glass were co-stained with anti-cav-1 (red) and anti-parkin (green), and observed by confocal microscopy. Blue indicates DAPI staining. Scale bar indicates 10 μm. c An in situ PLA assay was performed in WT MEF cells, as described in ‘Methods’. Red PLA spots represent interactions between parkin and cav-1. Blue indicates DAPI staining. Scale bar indicates 10 μm. d Parkin KO MEF cells were transfected with the indicated combinations of plasmids for flag-parkin, His-ubiquitin (His-Ub), and cav-1-EGFP for 48 h, and were then incubated with 10 μM MG132 for 3.5 h. The lysates were immunoprecipitated with an anti-EGFP antibody, followed by Western blotting. e Parkin KO MEF cells were transfected with plasmids for flag-tagged WT parkin and mutants (T240R and T415N) for 48 h, and the lysates were then analyzed by SDS-PAGE and Western blotting. The band intensity of three independent experiments was quantified. P values were determined using one way ANOVA. ** p < 0.01