Figure 4. Hipk2−/− MEFs exhibit higher Parkin protein levels in the cytosol and mitochondria. A–H, Immunofluorescent confocal microscopic images highlight the relatively higher Parkin protein levels in control DMSO-treated and CCCP-treated Hipk2−/− MEFs. Both Hipk2+/+ and Hipk2−/− MEFs were transfected with constructs expressing FLAG-Parkin, treated with DMSO or CCCP (5 μm) for 2 h, fixed in 4% PFA for immunostaining using anti-FLAG and anti-Tom20 antibodies, and processed for image analyses using the Nikon C2 Confocal Microscope. Insets in A, C, E, and G represent higher-magnification images of the highlighted areas. Scale bars: A, C, E, G, 10 μm; insets, 2.5 μm. FLAG-Parkin and Tom20 immunofluorescent intensities were measured using Nikon NIS-Elements software by drawing a line across the cytoplasm of Hipk2+/+ and Hipk2−/− MEFs (white lines in the “merge” in A, C, E, and G). The signal intensity of Parkin and Tom20 and the extent of their colocalization were presented in the corresponding panels in B, D, F, and H, where arrowheads indicate the colocalization of Parkin and Tom20. I, Quantification of overall Parkin immunofluorescent signal intensity in Hipk2+/+ and Hipk2−/− MEFs (A, C, E, and G). Data represented the mean ± SEM from 28 to 32 Parkin-expressing Hipk2+/+ and Hipk2−/− MEFs from four independent biological replicates. Statistical analyses used the Student's t test: ****p < 0.001. J, Western blot analysis of Parkin protein levels, detected by anti-FLAG antibody, in the cytosol and mitochondria of Hipk2+/+ and Hipk2−/− MEFs before and after CCCP treatment. Antibodies for actin and Tom20 were used as loading controls for cytosolic and mitochondrial fractions, respectively. K, Quantification of Parkin protein levels in the cytosol and mitochondria of Hipk2+/+ and Hipk2−/− MEFs before and after CCCP treatment. Data represented the mean ± SEM from three independent biological replicates. Statistical analyses used the Student's t test: *p < 0.05 and **p < 0.01.