Fig. 2.

SLP-2 preferentially binds to the Parkin RING0 domain. A Overview of the Parkin fragments cloned in PARK5-HA mammalian expression vector. B Whole cell lysates of SH-SY5Y cells transfected with wild type (WT), C-terminal (C), or N-terminal (N) Parkin-HA and myc-tagged SLP-2 were subjected to co-IP using an anti-HA antibody, followed by Western blot analysis (WB) of input and IP fractions with the indicated antibodies. Statistical differences were calculated by one-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons ***p ≤ 0.001, ****p ≤ 0.0001. C Whole cell lysates of SH-SY5Y cells transfected with UBL-, RING0-, RING1-, or RING2-HA and SLP-2-myc were subjected to co-IP using an anti-HA antibody, followed by WB analysis of input and IP fractions with the indicated antibodies. Statistical differences were calculated by one-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons. **p ≤ 0.01, ***p ≤ 0.001. D Co-stainings of a PLA experiment for Parkin RING0-HA and SLP-2 with a mitochondrial and E a lysosomal marker show that the interaction signal co-localizes with the mitochondria but not with the lysosomes. Scale bar: 20 µm (D); scale bar: 10 µm (E)