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. 2022 Nov 12;298(12):102704. doi: 10.1016/j.jbc.2022.102704

Figure 2.

Figure 2

RING1 domain is essential for Parkin–PHB2 interaction.A, schematic diagram of Parkin constructs used in this study. B, after purification by GST binding resin, different constructs of GST-Parkin (as indicated) were separately mixed with His-PHB2. Bound proteins were analyzed by immunoblot analysis with anti-GST and anti-PHB2 antibodies, n = 3. C, different constructs of EGFP-Parkin and FLAG-PHB2 were transfected into HEK293T cells, and then they were treated with 5 μg/ml A/O for 4 h. Cell lysates were processed for immunoprecipitation analysis using anti-GFP antibody and then were analyzed by immunoblot with indicated antibodies, n = 3. D, HEK293 cells expressing EGFP vector or EGFP-Parkin (WT, ΔRING1-IBR- or ΔRING1 mutant) were treated with 5 μg/ml A/O for 4 h, and then, the cells were fixed and stained with anti-GFP and anti-PHB2 antibodies in PLA experiment. The cells were visualized using Nikon microscope. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bar, 10 μm. E, Violin plot of PLA puncta/cell in (D), n = 84, ∗∗p < 0.01, One-way ANOVA followed by post hoc Tukey’s tests. The gray dotted line represents the median, and the black dotted line represents the quartile. EGFP, enhanced green fluorescent protein; PLA, proximity ligation assay; PHB2, prohibitin 2.