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

Figure 6.

Figure 6

Parkin-mediated ubiquitination of PHB2 ensures efficient mitophagy.A, HEK293 cells were transfected with control or si-PHB2 and then were retransfected with mt-mKeima, FLAG-PHB2 (mock, WT or KR mutant) with or without EGFP-Parkin. After treated with 5 μg/ml A/O for 4 h, the cells were analyzed by confocal microscopy, and the cartoon model on the right elucidated the relative levels of mitophagy flux. Scale bars, 10 μm. B, quantification of 561 nm versus 458 nm channel signal in figure (A). Data from 18 cells from three independent experiments were represented as means ± S.D., ∗∗p < 0.01. One-way ANOVA followed by post hoc Tukey’s tests. C, HEK293T cells were transfected with control or si-PHB2 and then were re-transfected with mCherry-Parkin and FLAG-PHB2 (mock, WT or KR mutant). Then the cells were treated with DMSO or 1 μg/ml A/O for 24 h, and cell lysates were analyzed by immunoblot analysis using anti-HSP60, anti-TOM20, anti-PHB2 (for detecting endogenous PHB2), anti-FLAG (for detecting exogenous PHB2), and anti-Tubulin antibodies. D, HEK293 cells with si-PHB2 were re-transfected with mCherry-Parkin and FLAG-PHB2 (mock, WT, or KR mutant), followed by treatment with DMSO, 5 μg/ml A/O for 6 h or 1 μg/ml A/O for 24 h. The cells were fixed and stained with anti-TOM20 and anti-FLAG antibodies and then were analyzed by confocal microscopy. The dashed white line in 24 h A/O treatment groups is the exogenous Parkin-expression cells outline. Scale bars, 10 μm. E, HSP60 and TOM20 protein levels in cells with 24 h A/O treatment in figure (C) were normalized to Tubulin, compared with nontreated group. Data were collected from three independent experiments and were represented as means ± S.D., ns, not significantly different; ∗∗p < 0.01, t test. F, quantification of the percent of cells with reduced TOM20 and PHB2 in figure (D). Data collected from three independent experiments were represented as means ± S.D., ns, not significantly different; ∗∗p < 0.01, One-way ANOVA followed by post hoc Tukey’s tests. A/O, Antimycin A and Oligomycin A; EGFP, enhanced green fluorescent protein; PHB2, prohibitin 2; TOM20, translocase of outer mitochondrial membrane 20.