Fig 1: Pathogenic mutations in Parkin RING0 interfere with binding to SLP-2. A Representation of the three-dimensional structure of human Parkin showing the Parkin domains. B Whole cell lysates of SH-SY5Y cells transfected with RING0 Parkin-HA and three mutant RING0 fragments (P153A, K161A and K211A) 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. C HeLa cells were processed using PLA after transfection with RING0-HA and the mutant RING0 domains using anti-HA and anti-SLP-2 antibodies. For all three mutant RING0 fragments with one of the three missense mutations, a decreased interaction was detected. The PLA signal is visualized in red, DAPI-stained nuclei are shown in blue. Scale bar: 50 µm. Statistical differences were calculated by one-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons *p ≤ 0.05, **p ≤ 0.01
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)
Fig 3: Parkin and SLP-2 interact in human control fibroblasts and hiPSC-derived neurons. A Representative PLA staining for fibroblasts of a control individual under normal culture conditions and after CCCP treatment (3 h, 10 µM). The PLA signal is visualized in red, DAPI-stained nuclei are shown in blue. Scale bar: 10 µm. Quantification of PLA dots per cell shows a significantly higher PLA signal after CCCP exposure, indicating an increased interaction between the two proteins. Statistical differences were calculated by unpaired Mann Whitney U test ****p ≤ 0.0001. B hiPSC-derived neurons of a control individual were processed using PLA under normal culture conditions and after CCCP treatment (3 h, 10 µM). Scale bar: 10 µm. Quantification of PLA dots per cell shows a significantly higher PLA signal after CCCP exposure. Statistical differences were calculated by unpaired Mann Whitney U test. **p ≤ 0.005. The specificity of the PLA interaction results was confirmed by performing the experiments with only one of the two primary antibodies. C Far-Western blot analysis using recombinant SLP-2 (aa 41–356) and Parkin proteins (aa 1–465 and aa 138–465) shows the direct binding of the two proteins. D Quantification of PLA dots per cell in control and PRKN mutant fibroblast lines under normal culture conditions and after CCCP treatment (3 h, 10 µM) using an antibody directed against the Parkin N-terminus and an anti-SLP-2 antibody. Only in the control lines, CCCP treatment resulted in a significantly increased interaction. Statistical differences were calculated by one-way ANOVA followed by Tukey’s post hoc test to correct for multiple comparisons. * ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001
Supplier Page from Abcam for Anti-SLP-2 antibody