Journal: Cell Death & Disease

Article Title: Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson’s disease

doi: 10.1038/s41419-018-1154-0

Figure Lengend Snippet: a Representative immunoblots of TIM23 and TOM20 protein levels in substantia nigra homogenates from Ctrl ( n = 4–5) and PD patients ( n = 9). Protein levels were normalized relative to Ponceau-S. Quantification is depicted as fold change to Ctrl (* P < 0.05 after unpaired two-sided Student’s t-test). b ) Representative immunoblots of NDUFS3, COX IV and α-tubulin protein levels in substantia nigra homogenates from control (Ctrl, n = 5) and PD patients ( n = 9). Protein levels were normalized relative to α-tubulin (* P < 0.05 after unpaired two-sided Student’s t -test). Error bars indicate s.e.m.

Article Snippet: Human TIM23 and TOM20 plasmids cloned into a pCMV6-XL5 vector were obtained from Origene (Origene Technologies), respectively.

Techniques: Western Blot, Control

Journal: Cell Death & Disease

Article Title: Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson’s disease

doi: 10.1038/s41419-018-1154-0

Figure Lengend Snippet: a–c , e BE(2)-M17 cells were treated with MPP + (0.25, 0.5 or 1 mM as stated) or untreated (UT) for 24 h. a Representative immunoblots of TIM23, TOM20 and VDAC protein levels normalized relative to VDAC ( n = 3 independent experiments). Quantification is depicted as fold change to UT condition (* P < 0.05, ** P < 0.01 after one-way ANOVA test followed by Tukey’s post hoc test). b Representative images of mitoGFP-transfected BE(2)-M17 cells. Quantification is depicted as the fold change of mitoGFP intensity per cell compared with UT condition. A minimum of 35 cells were analyzed per condition (* P < 0.05 after unpaired two-sided Student’s t -test). Scale bar: 10 μm. c Representative immunoblots of GFP and β-actin protein levels. mitoGFP import was calculated as the ratio of mature (m) GFP compared to the sum of precursor (p) and mature GFP (total GFP; n = 5 independent experiments, *** P < 0.001 after unpaired two-sided Student’s t -test). d Representative immunoblots of radiolabelled OTC in isolated mitochondria UT or treated with MPP + (1 mM) at different time points. pOTC import was calculated as the percentage of mature (m) OTC compared to the sum of precursor (p) and mature OTC ( n = 3 independent experiments, *** P < 0.001 after unpaired two-sided Mann–Whitney test). e Representative images of NDUFS3 and COX IV immunofluorescence. Quantification is represented as the fold change in NDUFS3 or COX IV intensity compared with UT condition. A minimum of 35 cells were analyzed per condition (* P < 0.05, ** P < 0.01 after unpaired two-sided Student’s t -test or unpaired two-sided Mann–Whitney test). Scale bar: 10 μm. Error bars indicate s.e.m.

Article Snippet: Human TIM23 and TOM20 plasmids cloned into a pCMV6-XL5 vector were obtained from Origene (Origene Technologies), respectively.

Techniques: Western Blot, Transfection, Isolation, MANN-WHITNEY, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson’s disease

doi: 10.1038/s41419-018-1154-0

Figure Lengend Snippet: Vehicle- or pCMV6-XL5-TIM23-transfected (TIM23) BE(2)-M17 cells were untreated (UT) or treated with a – c 1 mM MPP + for 24 h or d 5 mM for 24 h. a Representative images of COX IV and TIM23 immunofluorescence. Quantification is depicted as the fold change in COX IV intensity compared with vehicle-transfected UT condition. Dashed line shows TIM23-overexpressing cells. In all, 15 to 60 cells were analyzed per condition (* P < 0.05, *** P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 10 μm. b ROS production measured as CM-H 2 DCFDA fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition ( n = 3 independent experiments, ** P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). c Mitochondrial membrane potential measured as TMRM fluorescence intensity. Quantification is depicted as the fold change in fluorescence intensity compared with vehicle-transfected UT condition ( n = 5 independent experiments, ** P < 0.01 after one-way ANOVA followed by Tukey’s post hoc test). d Cell death measured as propidium iodide (PI)-positive cells ( n = 5 independent experiments). Quantification is depicted as the fold change in the percentage of PI-positive cells compared with each control condition (** P < 0.01 after unpaired two-sided Student’s t -test). Error bars indicate s.e.m.

Article Snippet: Human TIM23 and TOM20 plasmids cloned into a pCMV6-XL5 vector were obtained from Origene (Origene Technologies), respectively.

Techniques: Transfection, Immunofluorescence, Fluorescence, Membrane, Control

Journal: Cell Death & Disease

Article Title: Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson’s disease

doi: 10.1038/s41419-018-1154-0

Figure Lengend Snippet: a Representative immunoblots of TIM23, TOM20 and VDAC protein levels in ventral midbrain of vehicle- (Veh, n = 5) and MPTP-treated ( n = 6–7) mice euthanized at different time points. Protein levels were normalized relative to VDAC and quantification is depicted as fold change to vehicle condition (* P < 0.05, *** P < 0.001 after one-way ANOVA followed by Tukey’s post hoc test). b Representative images of tyrosine hydroxylase (TH) and COX IV immunostained in ventral midbrain sections from vehicle- ( n = 5) or MPTP-treated ( n = 5) mice euthanized at day 2 after the last injection. Quantification is depicted as the fold change of COX IV intensity in TH-positive neurons compared to vehicle condition. White arrows indicate representative neurons. A minimum of 36 neurons were analyzed per animal (* P < 0.05 after unpaired two-sided Student’s t -test). Error bars indicate s.e.m.

Article Snippet: Human TIM23 and TOM20 plasmids cloned into a pCMV6-XL5 vector were obtained from Origene (Origene Technologies), respectively.

Techniques: Western Blot, Injection

Journal: Cell Death & Disease

Article Title: Defective mitochondrial protein import contributes to complex I-induced mitochondrial dysfunction and neurodegeneration in Parkinson’s disease

doi: 10.1038/s41419-018-1154-0

Figure Lengend Snippet: a Representative images of TIM23 and TH immunofluorescence in ventral midbrain sections from AAV-hTIM23 mice ( n = 3). Scale bar: 150 μm. b – d Sham- and AAV-hTIM23-injected mice were treated with vehicle or MPTP. b Representative images of TH and COX IV immunofluorescence in ventral midbrain sections (Sham-Vehicle n = 5; Sham-MPTP n = 6; AAV-hTIM23-Vehicle n = 5; AAV-hTIM23-MPTP n = 6). Quantification is depicted as the fold change in COX IV intensity compared with sham-injected vehicle-treated group (* P < 0.05, *** P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 50 μm. c Representative photomicrographs of TH immunohistochemistry in striatum (Sham-Vehicle n = 7; Sham-MPTP n = 5; AAV-hTIM23-Vehicle n = 8; AAV-hTIM23-MPTP n = 8). Quantification is depicted as the optical densitometry of striatal TH immunoreactivity in the different experimental groups at day 21 post MPTP (** P < 0.01 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. d Representative photomicrographs of TH immunohistochemistry SNpc (Sham-Vehicle n = 14; Sham-MPTP n = 10; AAV-hTIM23-Vehicle n = 7; AAV-hTIM23-MPTP n = 8). Quantification is depicted as the stereological cell counts of SNpc TH-immunoreactive neurons in the different experimental groups at day 21 post MPTP (* P < 0.05, *** P < 0.001 after two-way ANOVA followed by Tukey’s post hoc test). Scale bar: 500 μm. Error bars indicate s.e.m.

Article Snippet: Human TIM23 and TOM20 plasmids cloned into a pCMV6-XL5 vector were obtained from Origene (Origene Technologies), respectively.

Techniques: Immunofluorescence, Injection, Immunohistochemistry

Journal: medRxiv

Article Title: ME/CFS and PASC Patient-Derived Immunoglobulin Complexes Disrupt Mitochondrial Function and Alter Inflammatory Marker Secretion

doi: 10.1101/2025.08.06.25332978

Figure Lengend Snippet: IgG from ME/CFS patients induces mitochondrial fragmentation. A. Representative confocal microscopy images show mitochondrial architecture in primary HUVECs expressing stable GFP within mitochondria and exposed to 1⍰μg/mL purified IgG from ME/CFS, PCS-CFS, and MS patient and control sera. One representative image is shown for each condition. B. Quantification of average mitochondrial surface area in primary HUVECs exposed to 1⍰μg/mL purified IgG from ME/CFS (n=39), PCS-CFS (n=15), MS (n=20) patients, and healthy controls (n=41). At least 10 cells were imaged for each sample derived from 3 replicates. In the left panel, each point represents the mean surface area across all images for each subject. In the right panel, each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Left) HD vs ME/CFS, ***P=0.0004. HD vs PCS-CFS, *P=0164. HD vs MS, ns P=0.8676. (Right) HD vs ME/CFS, ****P<0.0001. HD vs PCS-CFS, **P=0.0067. HD vs MS, ns P=0.4794. C. Gender-based separation of average mitochondrial surface area from the above experiment. Each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Female) HD vs ME/CFS, **P=0.0018. HD vs PCS-CFS, *P=0173. HD vs MS, ns P=0.4938. (Male) HD vs ME/CFS, ns P=0.0976. HD vs PCS-CFS, ns P=0.5058. HD vs MS, ns P=0.4462. D. Capillary-based automated Immunoblot analysis of Mitofilin, Drp1, TIMM23, PLD6, Miga, LC3b, Pink1 protein levels in HUVEC cells 36 hours after exposure to 1μg/mL of purified IgG from ME/CFS (n=12), PCS-CFS (n=12), and MS (n=11) patient and control (n=12) sera. Vinculin was used as a loading control. E. Fold change values from the above immunoblot analysis were derived from densitometric analysis of bands, normalized to the same value for vinculin. Two-tailed Mann-Whitney U test. (Drp1) HD vs ME/CFS, ns P=0.0684. HD vs PCS-CFS, *P=0387. HD vs MS, ns P=0.7859. (PLD6) HD vs ME/CFS, ****P<0.0001. HD vs PCS-CFS, ***P=0.0007. HD vs MS, ns P=0.6075. (Timm23) HD vs ME/CFS, ns P=0.1402. HD vs PCS-CFS, *P=0.0317. HD vs MS, *P=0.0106. (Miga1) HD vs ME/CFS, *P=0.0284. HD vs PCS-CFS, *P=0.0449. HD vs MS, ns P=0.1896. (LC3b) HD vs ME/CFS, ns P=0.0684. HD vs PCS-CFS, ns P=0.1600. HD vs MS, ns P=0.6075. (Mitofilin) HD vs ME/CFS, **P=0.0056. HD vs PCS-CFS, **P=0.0029. HD vs MS, ns P=0.3164. F. Immunoblot analysis shows a decreased amount of intracellular IgG heavy and light chains in HUVECs pretreated with Fc blocker before the exposure to 1⍰μg/mL purified IgG from ME/CFS patients or control sera for 12⍰h. Vinculin was used as a loading control. G. Schematics of IgG cleavage assay. H. Immunoblot analysis shows the presence of intracellular IgG heavy and light chains in HUVECs after exposure to 1⍰μg/mL purified IgG, purified Fab fragments, and Fc fragments from ME/CFS patients or control sera for 12⍰h. Vinculin was used as a loading control. Fc-specific secondary antibody was used (second panel) to detect the full-length heavy chain of IgG and cleaved Fc fragments. I. Quantification of average mitochondrial surface area in primary HUVECs exposed to 1⍰μg/mL purified IgG or Fab Fragment or Fc fragment from 5 ME/CFS, and 5 controls. In the upper panel, each point represents the mean surface area across all images for each individual. In the lower panel, each point represents the surface area measured from a single image. Two-tailed Mann-Whitney U test. (Upper Panel) HD vs ME/CFS IgG, *P=0.0317. HD vs ME/CFS Fab, ns P=0.8413. HD vs ME/CFS Fc, ns P=0.6905. (Lower Panel) HD vs ME/CFS IgG, *P=0.0130. HD vs ME/CFS Fab, ns P=0.5572. HD vs ME/CFS Fc, ns P=0.3662.

Article Snippet: Immunoblotting was carried out as described before ( , ) using antibodies: Drp1 antibody (#sc-101270, Santa Cruz Biotechnology), Mfn1 antibody (#sc-166644, Santa Cruz Biotechnology), Mfn2 antibody (#sc-515647, Santa Cruz Biotechnology), PLD6 antibody (#ab237612, Abcam), FAM73A Antibody (#PA553611, Invitrogen), p53 antibody (#sc-126, Santa Cruz Biotechnology), TOMM20 antibody (#sc-17764, Santa Cruz Biotechnology), TIMM23 antibody (#sc-514463, Santa Cruz Biotechnology), LC3β antibody (#sc-376404, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Expressing, Purification, Control, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Western Blot, Cleavage Assay

Journal: Nucleic acids research

Article Title: DNA damage response regulator ATR licenses PINK1-mediated mitophagy.

doi: 10.1093/nar/gkaf178

Figure Lengend Snippet: Figure 3. ATR docks at mitochondria by direct interaction with PINK1 and TOM / TIM. ( A ) PLAs of ATR interaction with PINK1, TIM23, and TOM40 in U2OS cells without (shNC) or with ATR-KD (shATR). Left panel: Representative images of PLA-positive signals (red foci) after co-staining with mitochondrial marker MitoTracker. Yellow arrows indicate PLA foci in mitochondria. Right panel: Quantification of PLA-positive foci from > 200 cells scored. Statistics were performed using a two-way ANO V A with a Tukey’s multiple comparisons test. n = 3. ( B ) Co-IPs by the FLAG antibody in FLAG-ATR and HA-TOM40 transfected HEK239T cells f ollo w ed b y w estern blot analy sis of the indicated proteins. β-actin w as used to control protein loading. n = 3. ( C ) Co-IPs by the FLAG antibody in FLAG-ATR and HA-TIM23 transfected HCT116 cells f ollo w ed b y w estern blot analy sis of the indicated proteins. β-actin was used to control protein loading. n = 3. ( D ) HCT116 cells with targeted siRNA against ATR (siATR), PINK1 (siPINK1), HSP90 α (siHSP90), TIM23 (siTIM23), and TOM7 (siTOM7) are analyzed by Western blotting for the indicated proteins in mitochondrial fractionations. ATP5A was used to control protein loading. n = 3. ( E ) 1D BN–PAGE analysis of ATR, TOM22, and PINK1 from the purified mitochondrial fraction of N2A cells. Under native conditions, ATR, TOM22, and PINK1 migrate at the same speed in native PAGE gel. After heat treatment, denatured samples were pulled down by an anti-ATR ab. The native and denatured samples were blotted with the indicated antibodies, which detect the respective proteins in denatured gels (lane “heat denaturing +”). n = 3.

Article Snippet: Scrambled siRNAs (siCtl: equal mixture of sc-37007, c-44230, and sc-44231) and targeted siRNAs against ATR siATR#1: sc-29763), PINK1 (siPINK1#1: sc-44598), TIM23 sc-44155), TOM7 (sc-89537), and HSP90 (sc-35608) were urchased from Santa Cruz Biotechnologies, and (siATR#2: HU040341-50UG) (siATR mix: equal mixture of siATR#1 nd #2) and (siPINK1#2: EHU057101-5OUG) (siPINK1 mix: qual mixture of siPINK1#1 and #2) from Sigma–Aldrich.

Techniques: Staining, Marker, Transfection, Control, Western Blot, Purification, Clear Native PAGE

Journal: EMBO Molecular Medicine

Article Title: Parkin is a disease modifier in the mutant SOD 1 mouse model of ALS

doi: 10.15252/emmm.201808888

Figure Lengend Snippet: A, B Representative Western blot (A) and quantification (B) of p62 associated with mitochondria from spinal cords. Protein levels are normalized by subunit ATPase β of mitochondria (Complex V). Mitochondrial p62 levels are increased at 120 days (symptomatic stage) in SOD1‐G93A spinal cord relative to Non Tg and wild‐type SOD1 (wtSOD1). Results are expressed as mean ± SEM relative to Non Tg at 30 days; n = 8 mice (four males and four females). At 120 days, ** P = 0.0018 (Non Tg vs. SOD1‐G93A) and ** P = 0.0011 (SOD1‐G93A vs. wtSOD1) by paired one‐way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one‐way ANOVA with Tukey's correction at 60 and 90 days). C, D Representative Western blot (C) and quantification (D) of Tim23 in homogenates from spinal cords using β‐actin as normalizer. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n = 6 mice (three males and three females). *** P = 0.0002 (Non Tg vs. SOD1‐G93A at 120 days) by paired one‐way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one‐way ANOVA with Tukey's correction at 60 and 90 days). Tim23 is decreased in SOD1‐G93A spinal cord relative to Non Tg at 120 days. E, F Western blot (E) and quantification (F) of COXI in homogenates from spinal cords. Protein levels are normalized by β‐actin. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n = 6 mice (three males and three females). * P = 0.017 (Non Tg vs. SOD1‐G93A at 120 days) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 and 60 days and paired one‐way ANOVA with Tukey's correction at 90 days). COX1 is decreased in SOD1‐G93A spinal cord relative to Non Tg at 120 days. G qPCR (fold change) of PGC1α mRNA normalized by β‐actin mRNA. Results are expressed as mean ± SEM and fold change of Non Tg at 30 days; n = 6 (three males and three females) for 30 and 120 days, n = 4 (two males and two females) for 60 days and n = 5 (three males and two females) for 90 days. * P = 0.035 at 90 days and *** P = 0.0007 at 120 days. Paired Student's t ‐test (for 60, 90, and 120 days) and paired Wilcoxon's test (for 30 days) were used for comparisons. Source data are available online for this figure.

Article Snippet: The following antibodies were used in Western blot (additional information can be found in ): Parkin (clone PRK8, 1:1,000, cat. # sc‐32282, Santa Cruz Biotechnology; Dallas, TX), β‐actin (clone AC‐74, 1:1,000–1:5,000, cat. # A5316, Sigma), p62 (clone 2C11, 1:1,000, cat. # H00008878‐M01, Novus Biologicals; Littleton, CO), optineurin (OPTN, 1:1,000, cat. # 10837‐1‐AP, Proteintech; Rosemont, IL), Complex V (ATP synthase beta, clone 3D5AB1, 1:1,000–1:5,000, cat. # A21351, Thermo Fisher Scientific; Waltham, MA), Tim23 (clone 32/Tim23, 1:1,000, cat. # 611222, BD Transduction Labs; San Jose, CA), COX1 (Complex IV, subunit 1, clone 1D6E1A8, 1:2,000, cat. #459600, Thermo Fisher Scientific), SOD1 (1:1,000, cat. # 574597, EMD Millipore; Billerica, MA), VCP (valosin‐containing protein, clone 5, 1:1,000, cat. # MA3‐004, Thermo Scientific; Rockford, IL), LC3 (microtubule‐associated protein 1A/1B‐light chain 3B, LC3B, 1:1,000, cat. # L7543, Sigma), March5 (membrane‐associated ring‐CH‐type finger 5, 1:1,000, cat. # PA5‐25584, Thermo Fisher Scientific), Mul1 (mitochondrial E3‐ubiquitin protein ligase 1 1:1,000, cat. # HPA017681, Sigma), Miro1 (mitochondrial Rho GTPase 1, clone 4H4, 1:1,000, cat. # H00055288‐M01, Novus Biologicals), mitofusin 2 (Mfn2, 1:1,000, cat. # M6319, Sigma), PARIS (parkin‐interacting substrate (ZNF746), clone N196/16, 1:1,000, cat. # 75‐195, NeuroMabs and Antibodies Inc.; Davis, CA), ubiquitin Lys48‐specific (clone Apu2, 1:1,000, cat. # 05‐1307, EMD Millipore), ubiquitin Lys63‐specific (clone Apu3, 1:1,000, cat. # 05‐1308, EMD Millipore), and horseradish‐peroxidase (HRP) goat anti‐rabbit, goat anti‐mouse, and donkey anti‐sheep secondary antibodies (1:10,000, Jackson ImmunoResearch; West Groove, PA).

Techniques: Western Blot

Journal: EMBO Molecular Medicine

Article Title: Parkin is a disease modifier in the mutant SOD 1 mouse model of ALS

doi: 10.15252/emmm.201808888

Figure Lengend Snippet: A, B Western blots of COXI (A) and Tim23 (B) in spinal cord homogenates at 130 days. C Quantification of COXI at 130 days with β‐actin as a normalizer. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.493 by paired Student's t ‐test). D Quantification of Tim23 at 130 days, using β‐actin as loading control, showed decreased levels of Tim23 in G93A mice. Results are expressed as mean ± SEM and as a percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.921 by paired Student's t ‐test). * P = 0.035 (Non Tg Vs. G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found. E, F Representative Western blots of COXI (E) and Tim23 (F) in disease end‐stage homogenates. G Quantification of COXI at disease end stage using β‐actin as loading control. Results are expressed as mean ± SEM and as percent of Non Tg; n = 5 (three males and two females) mice per group. * P = 0.047 for (G93A and PKO/G93A) by paired Student's t ‐test; ** P = 0.0018 by paired Friedman's test with Dunn's correction (Non Tg vs. G93A). H Quantification of Tim23 with β‐actin as normalizer at end stage showed that Parkin knockout can alleviate the turnover of mitochondrial proteins in G93A spinal cords. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.190 by paired Student's t ‐test). * P = 0.037 (for PKO vs. PKO/G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found. Source data are available online for this figure.

Article Snippet: The following antibodies were used in Western blot (additional information can be found in ): Parkin (clone PRK8, 1:1,000, cat. # sc‐32282, Santa Cruz Biotechnology; Dallas, TX), β‐actin (clone AC‐74, 1:1,000–1:5,000, cat. # A5316, Sigma), p62 (clone 2C11, 1:1,000, cat. # H00008878‐M01, Novus Biologicals; Littleton, CO), optineurin (OPTN, 1:1,000, cat. # 10837‐1‐AP, Proteintech; Rosemont, IL), Complex V (ATP synthase beta, clone 3D5AB1, 1:1,000–1:5,000, cat. # A21351, Thermo Fisher Scientific; Waltham, MA), Tim23 (clone 32/Tim23, 1:1,000, cat. # 611222, BD Transduction Labs; San Jose, CA), COX1 (Complex IV, subunit 1, clone 1D6E1A8, 1:2,000, cat. #459600, Thermo Fisher Scientific), SOD1 (1:1,000, cat. # 574597, EMD Millipore; Billerica, MA), VCP (valosin‐containing protein, clone 5, 1:1,000, cat. # MA3‐004, Thermo Scientific; Rockford, IL), LC3 (microtubule‐associated protein 1A/1B‐light chain 3B, LC3B, 1:1,000, cat. # L7543, Sigma), March5 (membrane‐associated ring‐CH‐type finger 5, 1:1,000, cat. # PA5‐25584, Thermo Fisher Scientific), Mul1 (mitochondrial E3‐ubiquitin protein ligase 1 1:1,000, cat. # HPA017681, Sigma), Miro1 (mitochondrial Rho GTPase 1, clone 4H4, 1:1,000, cat. # H00055288‐M01, Novus Biologicals), mitofusin 2 (Mfn2, 1:1,000, cat. # M6319, Sigma), PARIS (parkin‐interacting substrate (ZNF746), clone N196/16, 1:1,000, cat. # 75‐195, NeuroMabs and Antibodies Inc.; Davis, CA), ubiquitin Lys48‐specific (clone Apu2, 1:1,000, cat. # 05‐1307, EMD Millipore), ubiquitin Lys63‐specific (clone Apu3, 1:1,000, cat. # 05‐1308, EMD Millipore), and horseradish‐peroxidase (HRP) goat anti‐rabbit, goat anti‐mouse, and donkey anti‐sheep secondary antibodies (1:10,000, Jackson ImmunoResearch; West Groove, PA).

Techniques: Western Blot, Knock-Out