Journal: Kidney international

Article Title: Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition.

doi: 10.1038/sj.ki.5002632

Figure Lengend Snippet: Figure 6 | In situ calibration of SNARF-1 in MDCK cells. Cells were loaded (loading Protocol 2) with SNARF-1 and MTG and then incubated in high K þ-buffer supplemented with nigericin (13 mM), FCCP (1 mM), and oligomycin (20 mg/ml). Averaged SNARF-1 ratios (680/590 nm)±s.e.m. for cytosol (J; N ¼ 5, n ¼ 48) and mitochondria (’; N ¼ 5, n ¼ 48) were plotted against the pH of the bathing solution.

Article Snippet: In situ calibration: In situ calibration of SNARF-1 was carried out in separate experiments using the method of Thomas et al.39 MDCK cells were loaded according to Protocol 2 with SNARF-1 in the cytosol and with SNARF-1 plus MTG in mitochondria and exposed to high-Kþ buffer (vide infra Solutions and Chemicals) containing also 13 mM nigericin, 1 mM FCCP, and 20 mg ml 1 oligomycin (Tocris, Bristol, UK).

Techniques: In Situ, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

doi: 10.1016/j.jbc.2022.102704

Figure Lengend Snippet: Parkin directly interacts with PHB2, and the interaction is enhanced during mitophagy. A and B , HEK293T cells expressing EGFP vector or EGFP-Parkin with FLAG vector or FLAG-PHB2 were treated with 5 μg/ml A/O (Antimycin A and Oligomycin A) for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-GFP antibody ( A ) or anti-IgG (R) antibody ( B ). Co-immunoprecipitated FLAG-PHB2 was detected using anti-FLAG antibody, n = 3. C – E , primary murine embryonic fibroblasts cells ( C ), SH-SY5Y cells (infected with lentivirus carrying mCherry-Parkin) ( D ) or HeLa cells ( E ) were treated with 5 μg/ml A/O for 4 h, and then cell lysates were subjected to immunoprecipitation analysis using anti-PHB2 antibody. Co-immunoprecipitated proteins were detected using anti-Parkin and anti-LC3 antibodies, n = 3. F–G , HEK293T cells expressing FLAG vector or FLAG-PHB2 with EGFP-Parkin were treated with DMSO or 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody ( F ) or anti-IgG (M) antibody ( G ), and co-immunoprecipitated EGFP-Parkin was detected with anti-GFP antibody, n = 3. H–I , after purification by GST ( H ) or His ( I ) binding resin, GST-tagged ( H ) or His-tagged ( I ) proteins were separately mixed with His-PHB2 ( H , left region ), His-mCherry ( H , right region ), GST-Parkin ( I , left region ), or GST ( I , right region ). 10% input was loaded as control. The bound proteins were analyzed by immunoblot analysis with anti-GST or anti-His antibodies, n = 3. EGFP, enhanced green fluorescent protein; PHB2, prohibitin 2.

Article Snippet: The following chemicals were used for treatment: Antimycin A (1 or 5 μg/ml, Sigma, A8674), oligomycin A (1 or 5 μg/ml, Selleck Chemicals, S1478), 100 nM bafilomycin A 1 (Sigma-Aldrich, 196000), cycloheximide (100 μg/ml, MedChemExpress, HY-12320), and MG132 (10 μM, Calbiochem, 474787).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Infection, Purification, Binding Assay, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

doi: 10.1016/j.jbc.2022.102704

Figure Lengend Snippet: Parkin-mediated ubiquitination enhances the association of PHB2 with LC3 upon proteasomal-driven OMM rupture. A and B , after purification by GST or His binding resin, purified GST-tagged ( A ) or His-tagged ( B ) proteins were mixed with His-PHB2 ( A ) or GST-LC3 ( B ). 10% input was loaded as control, and the bound proteins were analyzed by immunoblot analysis using anti-His and anti-GST antibodies, n = 3. C , HEK293T cells expressing EGFP vector or EGFP-LC3 with FLAG-PHB2 were treated with DMSO or 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-GFP antibody and then were analyzed by immunoblot with anti-FLAG and anti-GFP antibodies, n = 3. D , after purification by His binding resin, purified His-tagged proteins were mixed with GST-LC3, with or without different concentration of GST-Parkin. The bound proteins were analyzed by immunoblot analysis with anti-LC3, anti-Parkin and anti-His antibodies, n = 3. E , WT or E478G mutant HA-OPTN, WT or T240R mutant mCherry-Parkin, WT or KR mutant FLAG-PHB2 with EGFP-LC3 were transfected into si-OPTN HEK293T cells, followed by treatment with 5 μg/ml A/O for 4 h. Cell lysates were subjected to immunoprecipitation analysis using anti-FLAG antibody and then were analyzed by immunoblot with anti-FLAG and anti-GFP antibodies, n = 3. F , FLAG-PHB2 (WT or KR mutant) with or without HA-Parkin were transfected into HEK293 cells, followed by treatment with 5 μg/ml A/O for 4 h with or without MG132 (10 μM, pretreated for 8 h). Then the cells were fixed and stained with anti-FLAG and anti-LC3 antibodies in PLA experiment. The cells were visualized using Nikon microscope. Blue : nuclei (DAPI); white dots: PLA puncta. Scale bar, 10 μm. G , Violin plot of PLA puncta/cell in ( F ), 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. H , after transfected with mCherry-Parkin and treated with 5 μg/ml A/O for 4 h with or without the combination of MG132 (10 μM, pretreated for 8 h), HEK293T cells were collected for mitochondria extraction. Then the mitochondria were treated with or without 10 μg/ml proteinase K for 30 min on ice before subjected to immunoblot analysis using anti-COXIV, anti-TOM20, and anti-HSP60 antibodies, n = 3. I , primary cortical neurons were transfected with lentiviral mt-mKeima and treated with 5 μg/ml A/O for 2 h with or without MG132 (10 μM, pretreated for 10 h). Then, the cells were visualized using confocal microscopy. Scale bar, 10 μm. J , quantification of 561 nm versus 458 nm channel signal in figure ( I ). Data were collected from nine cells from three independent experiments and were represented as means ± S.D., ns, not significantly different; ∗ p < 0.05, t test. A/O, Antimycin A and Oligomycin A; EGFP, enhanced green fluorescent protein; OPTN: optineurin; PLA, proximity ligation assay; PHB2, prohibitin 2; TOM20, translocase of outer mitochondrial membrane 20.

Article Snippet: The following chemicals were used for treatment: Antimycin A (1 or 5 μg/ml, Sigma, A8674), oligomycin A (1 or 5 μg/ml, Selleck Chemicals, S1478), 100 nM bafilomycin A 1 (Sigma-Aldrich, 196000), cycloheximide (100 μg/ml, MedChemExpress, HY-12320), and MG132 (10 μM, Calbiochem, 474787).

Techniques: Ubiquitin Proteomics, Purification, Binding Assay, Control, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation, Concentration Assay, Mutagenesis, Transfection, Staining, Microscopy, Extraction, Confocal Microscopy, Proximity Ligation Assay, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

doi: 10.1016/j.jbc.2022.102704

Figure Lengend Snippet: Parkin-mediated ubiquitination of PHB2 is required for the recruitment of autophagosomes onto the mitochondria. A – H , HEK293 cells were transfected with control or si-PHB2 and then were re-transfected with FLAG-Parkin, BFP-Mito, EGFP-OPTN, and mCherry-LC3 with or without FLAG-PHB2 (WT or KR mutant). After treated with 5 μg/ml A/O for 6 h, the cells were analyzed by multichannel live cell imaging using confocal microscopy. Regions in white box were magnified on the right side. White arrows indicated that LC3 “rings” stably localized on the surface of damaged mitochondria at different times in ( A ) and ( E ). The fluorescence intensity of the paint line ( white line ) was measured in ( B , D , F , and H ). LC3 ( red arrow ) could surround the damaged mitochondrion ( blue arrow ) in ( B and F ), but not in ( D and H ). Scale bars, 2.5 μm. A/O, Antimycin A and Oligomycin A; EGFP, enhanced green fluorescent protein; OPTN: optineurin; PHB2, prohibitin 2; TOM20: translocase of outer mitochondrial membrane 20.

Article Snippet: The following chemicals were used for treatment: Antimycin A (1 or 5 μg/ml, Sigma, A8674), oligomycin A (1 or 5 μg/ml, Selleck Chemicals, S1478), 100 nM bafilomycin A 1 (Sigma-Aldrich, 196000), cycloheximide (100 μg/ml, MedChemExpress, HY-12320), and MG132 (10 μM, Calbiochem, 474787).

Techniques: Ubiquitin Proteomics, Transfection, Control, Mutagenesis, Live Cell Imaging, Confocal Microscopy, Stable Transfection, Fluorescence, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

doi: 10.1016/j.jbc.2022.102704

Figure Lengend Snippet: 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.

Article Snippet: The following chemicals were used for treatment: Antimycin A (1 or 5 μg/ml, Sigma, A8674), oligomycin A (1 or 5 μg/ml, Selleck Chemicals, S1478), 100 nM bafilomycin A 1 (Sigma-Aldrich, 196000), cycloheximide (100 μg/ml, MedChemExpress, HY-12320), and MG132 (10 μM, Calbiochem, 474787).

Techniques: Ubiquitin Proteomics, Transfection, Control, Mutagenesis, Confocal Microscopy, Western Blot, Staining, Expressing, Membrane