Fig. 5
Parkin regulates lipid rafts-dependent endocytosis via cav-1 in neurons. a Rat primary cortical neurons were infected with shRNA lentivirus for non-targeting (Sh-NT) and parkin (Sh-Parkin) 11 days after plating, and cultured for a further 3 days. The lysates were analyzed by SDS-PAGE and Western blotting. b An in situ PLA assay was performed in rat primary cortical neurons, as described in ‘Methods’. Red PLA spots represent interactions between parkin and cav-1. Blue indicates DAPI staining. Scale bar indicates 20 μm. c The total cholesterol level was measured, as described in ‘Methods’. P values were determined using One way ANOVA. *p < 0.05. d Rat primary cortical neurons were infected with shRNA lentivirus for non-targeting (Sh-NT) and parkin (Sh-Parkin), stained with 0.5 μM C-laurdan, and then observed by two-photon microscopy. Images were processed for obtaining GP values as described in ‘Methods’. Scale bar indicates 20 μm. e Rat primary cortical neurons were infected with shRNA lentivirus for non-targeting (Sh-NT) and parkin (Sh-Parkin) and incubated with 50 nM BOIPY® FL C5-Lactosylceramide The cells were then fixed and observed by confocal microscopy. Scale bar indicates 20 μm. The intensity of three independent experiments was quantified. The mean value of intensity of each experiment was acquired by measuring the intensity of at least five random fields. P values were determined using a Two way ANOVA. **p < 0.01