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timm22  (Proteintech)


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    Structured Review

    Proteintech timm22
    OCT3 exists in mitochondria and <t>TIMM22</t> knockdown reduces mitochondrial OCT3 levels. A Representative TEM images of immunolabeling for OCT3 in mitochondria of SH-SY5Y cells. Arrowheads indicate the positions of immunogold particles identifying OCT3 on inner mitochondrial membrane (IMM), outer mitochondrial membrane (OMM), or mitochondrial matrix. Scale bar = 100 nm. B Representative western blot images of TIMM22 and OCT3 in whole-cell lysates with or without TIMM22 knockdown. Quantification of TIMM22 ( C ) and OCT3 ( D ) levels in whole-cell lysates. n = 3 independent experiments. Representative western blot images ( E ) and quantification ( F ) of TIMM22, OCT3, ANT3, PiC, TIMM23, and ATP5B in mitochondria extracts. n = 4 independent experiments. Data were shown as mean ± SEM. Unpaired t -test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 vs ScrRNA. ns, no significance
    Timm22, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/timm22/TIM22+Antibody/pmc12261742-244-82-88
    Average 93 stars, based on 45 article reviews
    timm22 - by Bioz Stars, 2026-09
    93/100 stars

    Images

    1) Product Images from "Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner"

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    Journal: BMC Biology

    doi: 10.1186/s12915-025-02318-4

    OCT3 exists in mitochondria and TIMM22 knockdown reduces mitochondrial OCT3 levels. A Representative TEM images of immunolabeling for OCT3 in mitochondria of SH-SY5Y cells. Arrowheads indicate the positions of immunogold particles identifying OCT3 on inner mitochondrial membrane (IMM), outer mitochondrial membrane (OMM), or mitochondrial matrix. Scale bar = 100 nm. B Representative western blot images of TIMM22 and OCT3 in whole-cell lysates with or without TIMM22 knockdown. Quantification of TIMM22 ( C ) and OCT3 ( D ) levels in whole-cell lysates. n = 3 independent experiments. Representative western blot images ( E ) and quantification ( F ) of TIMM22, OCT3, ANT3, PiC, TIMM23, and ATP5B in mitochondria extracts. n = 4 independent experiments. Data were shown as mean ± SEM. Unpaired t -test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 vs ScrRNA. ns, no significance
    Figure Legend Snippet: OCT3 exists in mitochondria and TIMM22 knockdown reduces mitochondrial OCT3 levels. A Representative TEM images of immunolabeling for OCT3 in mitochondria of SH-SY5Y cells. Arrowheads indicate the positions of immunogold particles identifying OCT3 on inner mitochondrial membrane (IMM), outer mitochondrial membrane (OMM), or mitochondrial matrix. Scale bar = 100 nm. B Representative western blot images of TIMM22 and OCT3 in whole-cell lysates with or without TIMM22 knockdown. Quantification of TIMM22 ( C ) and OCT3 ( D ) levels in whole-cell lysates. n = 3 independent experiments. Representative western blot images ( E ) and quantification ( F ) of TIMM22, OCT3, ANT3, PiC, TIMM23, and ATP5B in mitochondria extracts. n = 4 independent experiments. Data were shown as mean ± SEM. Unpaired t -test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 vs ScrRNA. ns, no significance

    Techniques Used: Knockdown, Immunolabeling, Membrane, Western Blot

    Downregulation of TIMM22 ameliorates MPP + induced damage to mitochondrial morphology. A Representative TEM images showing mitochondrial morphology in SH-SY5Y cells after MPP + treatment with or without TIMM22 knockdown. Scale bar = 0.5 μm or 100 nm for magnified images. Quantitative analysis of mitochondrial morphology from different groups including B mitochondrial number, n = 100 visual fields per group, C damaged mitochondria proportion, n = 100 visual fields per group, D mitochondrial area, n = 100 mitochondria per group, E aspect ratio, n = 150 mitochondria per group, F circularity, n = 150 mitochondria per group, and G cristae number per mitochondria, n = 100 mitochondria per group. Data were obtained from 3 independent experiments and shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA+Veh
    Figure Legend Snippet: Downregulation of TIMM22 ameliorates MPP + induced damage to mitochondrial morphology. A Representative TEM images showing mitochondrial morphology in SH-SY5Y cells after MPP + treatment with or without TIMM22 knockdown. Scale bar = 0.5 μm or 100 nm for magnified images. Quantitative analysis of mitochondrial morphology from different groups including B mitochondrial number, n = 100 visual fields per group, C damaged mitochondria proportion, n = 100 visual fields per group, D mitochondrial area, n = 100 mitochondria per group, E aspect ratio, n = 150 mitochondria per group, F circularity, n = 150 mitochondria per group, and G cristae number per mitochondria, n = 100 mitochondria per group. Data were obtained from 3 independent experiments and shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA+Veh

    Techniques Used: Knockdown

    Downregulation of TIMM22 mitigates MPP + induced mitochondrial dysfunction. A Representative flow cytometry images of mitochondrial membrane potential (ΔΨm) measured by the JC-1 assay. The upper quadrant locates the cells with normal ΔΨm and the lower quadrant locates the cells with decreased ΔΨm. FCCP was used as a positive control. B Quantification of cells with decreased ΔΨm from different groups. n = 3 independent experiments. C Quantification of mitochondrial ROS levels from different groups. n = 4 independent experiments. D Quantified levels of MPP + uptake in whole cell and E mitochondria extracts under conditions of TIMM22 KD, OCT3 KD, or both TIMM22 KD and OCT3 KD. n = 3 independent experiments. F Quantification of complex I activity from different groups. n = 5 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis, followed by the Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh [vs ScrRNA in D and E ]; * P < 0.05, ** P < 0.01, *** P < 0.001 vs TIMM22 KD + MPP + [vs TIMM22 + OCT3 KD in D and E ]. ns, no significance vs ScrRNA + Veh
    Figure Legend Snippet: Downregulation of TIMM22 mitigates MPP + induced mitochondrial dysfunction. A Representative flow cytometry images of mitochondrial membrane potential (ΔΨm) measured by the JC-1 assay. The upper quadrant locates the cells with normal ΔΨm and the lower quadrant locates the cells with decreased ΔΨm. FCCP was used as a positive control. B Quantification of cells with decreased ΔΨm from different groups. n = 3 independent experiments. C Quantification of mitochondrial ROS levels from different groups. n = 4 independent experiments. D Quantified levels of MPP + uptake in whole cell and E mitochondria extracts under conditions of TIMM22 KD, OCT3 KD, or both TIMM22 KD and OCT3 KD. n = 3 independent experiments. F Quantification of complex I activity from different groups. n = 5 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis, followed by the Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh [vs ScrRNA in D and E ]; * P < 0.05, ** P < 0.01, *** P < 0.001 vs TIMM22 KD + MPP + [vs TIMM22 + OCT3 KD in D and E ]. ns, no significance vs ScrRNA + Veh

    Techniques Used: Flow Cytometry, Membrane, Positive Control, Activity Assay

    Downregulation of TIMM22 inhibits the BAX/BCL-xL-caspase 9/3 pathway stimulated by MPP + . A Representative western blot images of BAX, BCL-xL, caspase 9, and caspase 3, in whole-cell lysates from different groups. Quantified expression levels of B BAX and C BCL-xL in whole-cell lysates from different groups. D BAX/BCL-xL ratio calculated from western blot analysis. Quantified expression levels of E cleaved-caspase 9 and F caspase 3 in whole-cell lysates from different groups. n = 3–5 independent experiments. Enzyme activities of G caspase 9 and H caspase 3 from different groups and expressed as fold change over ScrRNA + Veh group. n = 3 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA + Veh
    Figure Legend Snippet: Downregulation of TIMM22 inhibits the BAX/BCL-xL-caspase 9/3 pathway stimulated by MPP + . A Representative western blot images of BAX, BCL-xL, caspase 9, and caspase 3, in whole-cell lysates from different groups. Quantified expression levels of B BAX and C BCL-xL in whole-cell lysates from different groups. D BAX/BCL-xL ratio calculated from western blot analysis. Quantified expression levels of E cleaved-caspase 9 and F caspase 3 in whole-cell lysates from different groups. n = 3–5 independent experiments. Enzyme activities of G caspase 9 and H caspase 3 from different groups and expressed as fold change over ScrRNA + Veh group. n = 3 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA + Veh

    Techniques Used: Western Blot, Expressing

    Schematic diagram summarizing the present study. OCT3 localizes on both the outer and inner mitochondrial membranes. TIM22, a critical translocator in the inner membrane protein transport system, regulates the levels of OCT3 in the membrane of mitochondria. OCT3 subsequently mediates the transport of MPP + to the mitochondrial matrix, leading to mitochondrial dysfunction and the activation of the apoptosis pathway. Knocking down TIMM22 reduces the levels of OCT3 on the mitochondria, which in turn decreases MPP + uptake and mitigates complex I inhibition. This protects the mitochondrial membrane potential (ΔΨm) and suppresses the downstream pathways involving ROS and the BAX/BCL-xL-caspase9/3 pathway
    Figure Legend Snippet: Schematic diagram summarizing the present study. OCT3 localizes on both the outer and inner mitochondrial membranes. TIM22, a critical translocator in the inner membrane protein transport system, regulates the levels of OCT3 in the membrane of mitochondria. OCT3 subsequently mediates the transport of MPP + to the mitochondrial matrix, leading to mitochondrial dysfunction and the activation of the apoptosis pathway. Knocking down TIMM22 reduces the levels of OCT3 on the mitochondria, which in turn decreases MPP + uptake and mitigates complex I inhibition. This protects the mitochondrial membrane potential (ΔΨm) and suppresses the downstream pathways involving ROS and the BAX/BCL-xL-caspase9/3 pathway

    Techniques Used: Membrane, Activation Assay, Inhibition

    Related Articles

    Western Blot:

    Article Title: Ovarian carcinoma immunoreactive antigen–like protein 2 (OCIAD2) is a novel complex III–specific assembly factor in mitochondria
    Article Snippet: .. The antibodies against the following proteins were used in the study: ALR (Santa Cruz Biotechnology; Sc-134869; 1:500), ATP5A (Abcam; ab14748; 1:500), ATP5B (rabbit serum; Rehling laboratory, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; 1:500), COA7 (Sigma; HPA029926; 1:500), COX1 (rabbit serum; Rehling laboratory; 1:2000), COX4 (Cell Signaling Technology; 4850; 1:2000), cytochrome B (rabbit serum; Rehling laboratory; 1:1000), HSP60 (Sigma; H4149; 1:500), MIC60 (Novus Biologicals; NB100-1919; 1:1000), MRPL1 (rabbit serum; Rehling laboratory; 1:1000), MRPL55 (Proteintech; 17679-1-AP; 1:500); MRPS18 (Proteintech; 16139-1-AP; 1:500), ND1 (rabbit serum; Rehling laboratory; 1:1000), ND2 (rabbit serum; Rehling laboratory; 1:1000), NDUFS1 (Santa Cruz Biotechnology; Sc-50132; 1:1000), prohibitin 2 (Sigma Aldrich; HPA039874; 1:1000), OCIAD2 (rabbit serum; Chacińska laboratory, IMol Polish Academy of Sciences, Warsaw, Poland; 1:1000), Rieske (rabbit serum; Rehling laboratory; 1:1000), TIMM22 (Proteintech; 14927-1-AP; 1:500), TIMM23 (BD Biosciences; 611222; 1:1000), TIMM29 (Proteintech; 25652-1-AP; 1:500), TOMM20 (Santa Cruz Biotechnology; Sc-11415; 1:500), tubulin (Santa Cruz Biotechnology; sc-134239; 1:2000), SDHA (Santa Cruz Biotechnology; Sc-166947; 1:1000), UQCC2 (Novus Biological; NBP2-14240; 1:500), UQCRB (Sigma Aldrich; HPA002815; 1:500), UQCR1 (Sigma; HPA002815; 1:500), Total OXPHOS Blue Native WB Antibody Cocktail (abcam; ab110412; 1:1000), OCIAD2 (Invitrogen; PA5-59375; 1:100). ..

    Article Title: Ovarian carcinoma immunoreactive antigen–like protein 2 (OCIAD2) is a novel complex III–specific assembly factor in mitochondria
    Article Snippet: .. The antibodies against the following proteins were used in the study: ALR (Santa Cruz Biotechnology; Sc-134869; 1:500), ATP5A (Abcam; ab14748; 1:500), ATP5B (rabbit serum; Rehling laboratory, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; 1:500), COA7 (Sigma; HPA029926; 1:500), COX1 (rabbit serum; Rehling laboratory; 1:2000), COX4 (Cell Signaling Technology; 4850; 1:2000), cytochrome B (rabbit serum; Rehling laboratory; 1:1000), HSP60 (Sigma; H4149; 1:500), MIC60 (Novus Biologicals; NB100-1919; 1:1000), MRPL1 (rabbit serum; Rehling laboratory; 1:1000), MRPL55 (Proteintech; 17679-1-AP; 1:500); MRPS18 (Proteintech; 16139-1-AP; 1:500), ND1 (rabbit serum; Rehling laboratory; 1:1000), ND2 (rabbit serum; Rehling laboratory; 1:1000), NDUFS1 (Santa Cruz Biotechnology; Sc-50132; 1:1000), prohibitin 2 (Sigma Aldrich; HPA039874; 1:1000), OCIAD2 (rabbit serum; Chacin ́ska laboratory, IMol Polish Academy of Sciences, Warsaw, Poland; 1:1000), Rieske (rabbit serum; Rehling laboratory; 1:1000), TIMM22 (Proteintech; 14927-1-AP; 1:500), TIMM23 (BD Biosciences; 611222; 1:1000), TIMM29 (Proteintech; 25652-1-AP; 1:500), TOMM20 (Santa Cruz Biotechnology; Sc-11415; 1:500), tubulin (Santa Cruz Biotechnology; sc-134239; 1:2000), SDHA (Santa Cruz Biotechnology; Sc-166947; 1:1000), UQCC2 (Novus Biological; NBP2-14240; 1:500), UQCRB (Sigma Aldrich; HPA002815; 1:500), UQCR1 (Sigma; HPA002815; 1:500), Total OXPHOS Blue Native WB Antibody Cocktail (abcam; ab110412; 1:1000), OCIAD2 (Invitrogen; PA5-59375; 1:100). ..

    FLAG-tag:

    Article Title: Cytosolic retention of the mitochondrial protease HtrA2 during mitochondrial protein import stress (MPIS) triggers the DELE1-HRI pathway
    Article Snippet: CCCP (C2759, Sigma), Puromycin (P8833, Sigma), MitoBloCK-6 (5.05759.0001, EMD Millipore), MitoBloCK-10 (HY-115467, MedChemExpress), FuGENE HD Transfection Reagent (E2311, Promega), Oligomycin A (75351, Sigma), Antimycin A (A8674, Sigma), MG132 (1748, Tocris), o -phenanthroline (516705, Sigma), AEBSF (A8456, Sigma) .. ATF4 (11815S, Cell Signaling, 1/2000), DELE1(PA5-57712, Thermo Fisher Scientific, 1/2000), HRI (20499-1-AP, Proteintech, 1/3000), GAPDH (5174S, Cell Signaling, 1/10000), HtrA2 (#9745, Cell Signaling, 1/1000), OMA1 (HPA055120, Sigma, 1/2500), OPA1 (612606, BD, 1/2500), NLRX1 (MA5-27207, Thermo Fisher Scientific, 1/3000), RRBP1 (PA5-21392, Thermo Fisher Scientific, 1/2000), Tubulin (T9026, Millipore Sigma, 1/10,000), VDAC1 (ab14734, abcam, 1/2000), COX4 (ab33985, abcam, 1/4000), LC3B (3868S, Cell Signaling, 1/1000, PINK1 (BC100-494, Novus, 1/1000), TOMM20 (42406, Cell Signaling, 1:2000), TOM40 (18409-1-AP, Proteintech, 1/10000), TOM70 (14528-1-AP, Proteintech, 1/10000), TIMM22 (14927-1-AP, Proteintech, 1/2500), MIA40 (21090-1-AP, Proteintech, 1/2500), TIMM23 (11123-1-AP, Proteintech, 1:2000), HA Tag (ab18181, abcam, 1/1000), FLAG Tag (14793S, Cell Signaling, 1/1000), HRP Rabbit (111-035-003, Jackson ImmunoResearch Laboratories, 1/10,000), HRP mouse (115-035-003, Jackson ImmunoResearch Laboratories, 1/10,000), Alexa fluor 488 rabbit (A11034, Life Technologies, 1/300), Cy3-conjugated goat anti-mouse (115-165-003, Jackson ImmunoResearch Laboratories, 1/300). ..

    Article Title: Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway
    Article Snippet: CCCP (C2759, Sigma), Puromycin (P8833, Sigma), MitoBloCK-6 (5.05759.0001, EMD Millipore), MitoBloCK-10 (HY-115467, MedChemExpress), FuGENE HD Transfection Reagent (E2311, Promega), Oligomycin A (75351, Sigma), Antimycin A (A8674, Sigma), MG132 (1748, Tocris), o -phenanthroline (516705, Sigma), AEBSF (A8456, Sigma), TMRE (T669, Thermo Fisher Scientific) and trans-Resveratrol (70675-50, Cederlane). .. ATF4 (11815S, Cell Signaling, 1/2000), DELE1(PA5-57712, Thermo Fisher Scientific, 1/2000), HRI (20499-1-AP, Proteintech, 1/3000), GAPDH (5174S, Cell Signaling, 1/10,000), HtrA2 (#9745, Cell Signaling, 1/1000), OMA1 (HPA055120, Sigma, 1/2500), OPA1 (612606, BD, 1/2500), NLRX1 (MA5-27207, Thermo Fisher Scientific, 1/3000), RRBP1 (PA5-21392, Thermo Fisher Scientific, 1/2000), Tubulin (T9026, Millipore Sigma, 1/10,000), VDAC1 (ab14734, abcam, 1/2000), COX4 (ab33985, abcam, 1/4000), LC3B 3868S, Cell Signaling, 1/1000, PINK1 (BC100-494, Novus, 1/1000), TOMM20 (42406, Cell Signaling, 1/2000), TOM40 (18409-1-AP, Proteintech, 1/10,000), TOM70 (14528-1-AP, Proteintech, 1/10,000), TIMM22 (14927-1-AP, Proteintech, 1/2500), MIA40 (21090-1-AP, Proteintech, 1/2500), TIMM23 (11123-1-AP, Proteintech, 1:2000), HA Tag (ab18181, abcam, 1/1000), FLAG Tag (14793S, Cell Signaling, 1/1000), LONP1 (15440-1-AP, Proteintech, 1/1000), Myc-tag (2276S, Cell Signaling, 1/1000), HRP Rabbit (111-035-003, Jackson ImmunoResearch Laboratories, 1/10,000), HRP mouse (115-035-003, Jackson ImmunoResearch Laboratories, 1/10,000), Alexa fluor 488 rabbit (A11034, Life Technologies, 1/300), Cy3-conjugated goat anti-mouse (115-165-003, Jackson ImmunoResearch Laboratories, 1/300). .. Note: We have been informed by Thermo Fisher Scientific, the supplier of the anti-DELE1 antibody that we used throughout the manuscript that this antibody was not available anymore, as the catalog number (PA5-57712) has been discontinued.

    other:

    Article Title: Ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) is a novel metazoan specific complex III assembly factor
    Article Snippet: The antibodies against following proteins were used in the study: ALR (Santa Cruz Biotechnology, Sc-134869, 1:500), ATP5A (Abcam, ab14748, 1:500), ATP5B (Rabbit serum, Rehling laboratory, 1:500), COA7 (Sigma, HPA029926, 1:500), COX1 (Rabbit serum, Rehling laboratory, 1:2000), COX4 (Cell Signaling Technology, 4850, 1:2000), Cytochrome B (Rabbit serum, Rehling laboratory, 1:1000), (HSP60 (Sigma, H4149, 1:500), MIC60 (Novus Biologicals, NB100-1919, 1:1000), MRPL1 (Rabbit serum, Rehling laboratory, 1:1000), MRPL55 (Proteintech, 17679-1-AP, 1:500), MRPS18(Proteintech 16139-1-AP, 1:500), ND1 (Rabbit serum, Rehling laboratory, 1:1000), ND2 (Rabbit serum, Rehling laboratory, 1:1000), NDUFS1 (Santa Cruz Biotechnology, Sc-50132, 1:1000), Prohibitin 2 (Sigma Aldrich, HPA039874, 1:1000), OCIAD2 (Rabbit serum, Chacińska laboratory, 1:1000), Prohibitin 2 (Sigma Rieske (Rabbit serum, Rehling laboratory, 1:1000), TIMM22 (Proteintech, 14927-1-AP, 1:500), TIMM23 (BD Biosciences, 611222, 1:1000), TIMM29 (Proteintech, 25652-1-AP, 1:500), TOMM20 (Santa Cruz Biotechnology, Sc-11415, 1:500), Tubulin (Santa Cruz Biotechnologys, sc-134239, 1:2000), SDHA (Santa Cruz Biotechnology, Sc-166947, 1:1,000), UQCC2 (Novus Biological, NBP2-14240, 1:500), UQCRB (Sigma Aldrich, HPA002815, 1:500),UQCR1 (Sigma, HPA002815, 1:500).

    Nucleic Acid Electrophoresis:

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1- AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.

    Blocking Assay:

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1- AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.

    Incubation:

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner.
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1- AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner
    Article Snippet: All cellular proteins were quantified by Pierce BCA Protein Assay Kit (Thermo; Cat. No. 23225), according to the manufacturer’s instructions. .. Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP). .. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Yeasen; Cat. No. 33101ES60) (1:5000, vol/vol) for 1 h at room temperature.



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    OCT3 exists in mitochondria and TIMM22 knockdown reduces mitochondrial OCT3 levels. A Representative TEM images of immunolabeling for OCT3 in mitochondria of SH-SY5Y cells. Arrowheads indicate the positions of immunogold particles identifying OCT3 on inner mitochondrial membrane (IMM), outer mitochondrial membrane (OMM), or mitochondrial matrix. Scale bar = 100 nm. B Representative western blot images of TIMM22 and OCT3 in whole-cell lysates with or without TIMM22 knockdown. Quantification of TIMM22 ( C ) and OCT3 ( D ) levels in whole-cell lysates. n = 3 independent experiments. Representative western blot images ( E ) and quantification ( F ) of TIMM22, OCT3, ANT3, PiC, TIMM23, and ATP5B in mitochondria extracts. n = 4 independent experiments. Data were shown as mean ± SEM. Unpaired t -test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 vs ScrRNA. ns, no significance

    Journal: BMC Biology

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    doi: 10.1186/s12915-025-02318-4

    Figure Lengend Snippet: OCT3 exists in mitochondria and TIMM22 knockdown reduces mitochondrial OCT3 levels. A Representative TEM images of immunolabeling for OCT3 in mitochondria of SH-SY5Y cells. Arrowheads indicate the positions of immunogold particles identifying OCT3 on inner mitochondrial membrane (IMM), outer mitochondrial membrane (OMM), or mitochondrial matrix. Scale bar = 100 nm. B Representative western blot images of TIMM22 and OCT3 in whole-cell lysates with or without TIMM22 knockdown. Quantification of TIMM22 ( C ) and OCT3 ( D ) levels in whole-cell lysates. n = 3 independent experiments. Representative western blot images ( E ) and quantification ( F ) of TIMM22, OCT3, ANT3, PiC, TIMM23, and ATP5B in mitochondria extracts. n = 4 independent experiments. Data were shown as mean ± SEM. Unpaired t -test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001 vs ScrRNA. ns, no significance

    Article Snippet: Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP).

    Techniques: Knockdown, Immunolabeling, Membrane, Western Blot

    Downregulation of TIMM22 ameliorates MPP + induced damage to mitochondrial morphology. A Representative TEM images showing mitochondrial morphology in SH-SY5Y cells after MPP + treatment with or without TIMM22 knockdown. Scale bar = 0.5 μm or 100 nm for magnified images. Quantitative analysis of mitochondrial morphology from different groups including B mitochondrial number, n = 100 visual fields per group, C damaged mitochondria proportion, n = 100 visual fields per group, D mitochondrial area, n = 100 mitochondria per group, E aspect ratio, n = 150 mitochondria per group, F circularity, n = 150 mitochondria per group, and G cristae number per mitochondria, n = 100 mitochondria per group. Data were obtained from 3 independent experiments and shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA+Veh

    Journal: BMC Biology

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    doi: 10.1186/s12915-025-02318-4

    Figure Lengend Snippet: Downregulation of TIMM22 ameliorates MPP + induced damage to mitochondrial morphology. A Representative TEM images showing mitochondrial morphology in SH-SY5Y cells after MPP + treatment with or without TIMM22 knockdown. Scale bar = 0.5 μm or 100 nm for magnified images. Quantitative analysis of mitochondrial morphology from different groups including B mitochondrial number, n = 100 visual fields per group, C damaged mitochondria proportion, n = 100 visual fields per group, D mitochondrial area, n = 100 mitochondria per group, E aspect ratio, n = 150 mitochondria per group, F circularity, n = 150 mitochondria per group, and G cristae number per mitochondria, n = 100 mitochondria per group. Data were obtained from 3 independent experiments and shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA+Veh

    Article Snippet: Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP).

    Techniques: Knockdown

    Downregulation of TIMM22 mitigates MPP + induced mitochondrial dysfunction. A Representative flow cytometry images of mitochondrial membrane potential (ΔΨm) measured by the JC-1 assay. The upper quadrant locates the cells with normal ΔΨm and the lower quadrant locates the cells with decreased ΔΨm. FCCP was used as a positive control. B Quantification of cells with decreased ΔΨm from different groups. n = 3 independent experiments. C Quantification of mitochondrial ROS levels from different groups. n = 4 independent experiments. D Quantified levels of MPP + uptake in whole cell and E mitochondria extracts under conditions of TIMM22 KD, OCT3 KD, or both TIMM22 KD and OCT3 KD. n = 3 independent experiments. F Quantification of complex I activity from different groups. n = 5 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis, followed by the Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh [vs ScrRNA in D and E ]; * P < 0.05, ** P < 0.01, *** P < 0.001 vs TIMM22 KD + MPP + [vs TIMM22 + OCT3 KD in D and E ]. ns, no significance vs ScrRNA + Veh

    Journal: BMC Biology

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    doi: 10.1186/s12915-025-02318-4

    Figure Lengend Snippet: Downregulation of TIMM22 mitigates MPP + induced mitochondrial dysfunction. A Representative flow cytometry images of mitochondrial membrane potential (ΔΨm) measured by the JC-1 assay. The upper quadrant locates the cells with normal ΔΨm and the lower quadrant locates the cells with decreased ΔΨm. FCCP was used as a positive control. B Quantification of cells with decreased ΔΨm from different groups. n = 3 independent experiments. C Quantification of mitochondrial ROS levels from different groups. n = 4 independent experiments. D Quantified levels of MPP + uptake in whole cell and E mitochondria extracts under conditions of TIMM22 KD, OCT3 KD, or both TIMM22 KD and OCT3 KD. n = 3 independent experiments. F Quantification of complex I activity from different groups. n = 5 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis, followed by the Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh [vs ScrRNA in D and E ]; * P < 0.05, ** P < 0.01, *** P < 0.001 vs TIMM22 KD + MPP + [vs TIMM22 + OCT3 KD in D and E ]. ns, no significance vs ScrRNA + Veh

    Article Snippet: Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP).

    Techniques: Flow Cytometry, Membrane, Positive Control, Activity Assay

    Downregulation of TIMM22 inhibits the BAX/BCL-xL-caspase 9/3 pathway stimulated by MPP + . A Representative western blot images of BAX, BCL-xL, caspase 9, and caspase 3, in whole-cell lysates from different groups. Quantified expression levels of B BAX and C BCL-xL in whole-cell lysates from different groups. D BAX/BCL-xL ratio calculated from western blot analysis. Quantified expression levels of E cleaved-caspase 9 and F caspase 3 in whole-cell lysates from different groups. n = 3–5 independent experiments. Enzyme activities of G caspase 9 and H caspase 3 from different groups and expressed as fold change over ScrRNA + Veh group. n = 3 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA + Veh

    Journal: BMC Biology

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    doi: 10.1186/s12915-025-02318-4

    Figure Lengend Snippet: Downregulation of TIMM22 inhibits the BAX/BCL-xL-caspase 9/3 pathway stimulated by MPP + . A Representative western blot images of BAX, BCL-xL, caspase 9, and caspase 3, in whole-cell lysates from different groups. Quantified expression levels of B BAX and C BCL-xL in whole-cell lysates from different groups. D BAX/BCL-xL ratio calculated from western blot analysis. Quantified expression levels of E cleaved-caspase 9 and F caspase 3 in whole-cell lysates from different groups. n = 3–5 independent experiments. Enzyme activities of G caspase 9 and H caspase 3 from different groups and expressed as fold change over ScrRNA + Veh group. n = 3 independent experiments. Data were shown as mean ± SEM. Two-way ANOVA was used for statistical analysis followed by Tukey's post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 vs ScrRNA + Veh; * P < 0.05, *** P < 0.001 vs TIMM22 KD + MPP + . ns, no significance vs ScrRNA + Veh

    Article Snippet: Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP).

    Techniques: Western Blot, Expressing

    Schematic diagram summarizing the present study. OCT3 localizes on both the outer and inner mitochondrial membranes. TIM22, a critical translocator in the inner membrane protein transport system, regulates the levels of OCT3 in the membrane of mitochondria. OCT3 subsequently mediates the transport of MPP + to the mitochondrial matrix, leading to mitochondrial dysfunction and the activation of the apoptosis pathway. Knocking down TIMM22 reduces the levels of OCT3 on the mitochondria, which in turn decreases MPP + uptake and mitigates complex I inhibition. This protects the mitochondrial membrane potential (ΔΨm) and suppresses the downstream pathways involving ROS and the BAX/BCL-xL-caspase9/3 pathway

    Journal: BMC Biology

    Article Title: Organic cation transporter 3 on neuronal mitochondria mediates MPP + -induced mitochondrial dysfunction and neurotoxicity in a TIMM22-dependent manner

    doi: 10.1186/s12915-025-02318-4

    Figure Lengend Snippet: Schematic diagram summarizing the present study. OCT3 localizes on both the outer and inner mitochondrial membranes. TIM22, a critical translocator in the inner membrane protein transport system, regulates the levels of OCT3 in the membrane of mitochondria. OCT3 subsequently mediates the transport of MPP + to the mitochondrial matrix, leading to mitochondrial dysfunction and the activation of the apoptosis pathway. Knocking down TIMM22 reduces the levels of OCT3 on the mitochondria, which in turn decreases MPP + uptake and mitigates complex I inhibition. This protects the mitochondrial membrane potential (ΔΨm) and suppresses the downstream pathways involving ROS and the BAX/BCL-xL-caspase9/3 pathway

    Article Snippet: Then, equal amounts of proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel (Beyotime; Cat. No. P0690) and transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes with a constant current of 250 mA for 90 min. After blocking with 5% wt/vol bovine serum albumin (BSA) for 1 h, the membranes were incubated at 4 °C overnight with primary antibodies of OCT3 (Affinity; Cat. No. DF4268), VDAC (Abcam; Cat. No. ab14734), calnexin (Abcam; Cat. No. ab227310), β-actin (CST; Cat. No. 4970), TIMM22 (Sangon; Cat. No. D225301), TIMM23 (Proteintech; Cat. No. 11123–1-AP), ATP5B (Proteintech; Cat. No. 17247–1-AP), ANT3 (Immunoway; Cat. No. YT0241), PiC (Proteintech; Cat. No. 10420–1-AP), caspase-9 (Proteintech; Cat. No. 10380-1AP), caspase-3 (CST; Cat. No. 9662), BAX (Proteintech; Cat. No. 50599–2-Ig), or BCL-xL (Proteintech; Cat No. 26967–1-AP).

    Techniques: Membrane, Activation Assay, Inhibition