sh-sy5y cells expressing p97 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore sh sy5y cells
    Inactivation of p97 impairs mitochondrial function during neurotoxic stress . (A) <t>SH-SY5Y</t> cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p
    Sh Sy5y Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1835 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sh sy5y cells/product/Millipore
    Average 99 stars, based on 1835 article reviews
    Price from $9.99 to $1999.99
    sh sy5y cells - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    94
    Millipore p97 inhibitor dbeq
    Inactivation of <t>p97</t> impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor <t>DBeQ</t> and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p
    P97 Inhibitor Dbeq, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p97 inhibitor dbeq/product/Millipore
    Average 94 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    p97 inhibitor dbeq - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    85
    rdi research diagnostics mouse monoclonal anti p97
    Inactivation of <t>p97</t> impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor <t>DBeQ</t> and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p
    Mouse Monoclonal Anti P97, supplied by rdi research diagnostics, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti p97/product/rdi research diagnostics
    Average 85 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti p97 - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    99
    Qiagen effectene transfection reagent
    Inactivation of <t>p97</t> impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor <t>DBeQ</t> and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p
    Effectene Transfection Reagent, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 13354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/effectene transfection reagent/product/Qiagen
    Average 99 stars, based on 13354 article reviews
    Price from $9.99 to $1999.99
    effectene transfection reagent - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    94
    Millipore nms 873
    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of <t>NMS‐873,</t> MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.
    Nms 873, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nms 873/product/Millipore
    Average 94 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    nms 873 - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    99
    Millipore l glutamine
    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of <t>NMS‐873,</t> MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.
    L Glutamine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1738 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l glutamine/product/Millipore
    Average 99 stars, based on 1738 article reviews
    Price from $9.99 to $1999.99
    l glutamine - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Millipore sodium pyruvate
    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of <t>NMS‐873,</t> MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.
    Sodium Pyruvate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sodium pyruvate/product/Millipore
    Average 99 stars, based on 13924 article reviews
    Price from $9.99 to $1999.99
    sodium pyruvate - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Millipore co2 incubator
    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of <t>NMS‐873,</t> MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.
    Co2 Incubator, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co2 incubator/product/Millipore
    Average 99 stars, based on 2037 article reviews
    Price from $9.99 to $1999.99
    co2 incubator - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    TaKaRa tet system approved fbs
    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of <t>NMS‐873,</t> MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.
    Tet System Approved Fbs, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tet system approved fbs/product/TaKaRa
    Average 99 stars, based on 850 article reviews
    Price from $9.99 to $1999.99
    tet system approved fbs - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Millipore mg132
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM <t>MG132</t> for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z‐VAD‐FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg132/product/Millipore
    Average 99 stars, based on 1045 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    93
    Peptide Institute z vad fmk
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Z Vad Fmk, supplied by Peptide Institute, used in various techniques. Bioz Stars score: 93/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/z vad fmk/product/Peptide Institute
    Average 93 stars, based on 152 article reviews
    Price from $9.99 to $1999.99
    z vad fmk - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    99
    Millipore glucose dmem
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Glucose Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glucose dmem/product/Millipore
    Average 99 stars, based on 836 article reviews
    Price from $9.99 to $1999.99
    glucose dmem - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Millipore fluorophore conjugated secondary antibodies
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Fluorophore Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorophore conjugated secondary antibodies/product/Millipore
    Average 99 stars, based on 130 article reviews
    Price from $9.99 to $1999.99
    fluorophore conjugated secondary antibodies - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Millipore rotenone
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Rotenone, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1894 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rotenone/product/Millipore
    Average 99 stars, based on 1894 article reviews
    Price from $9.99 to $1999.99
    rotenone - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    94
    Millipore amyloid β protein fragment 25 35
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Amyloid β Protein Fragment 25 35, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amyloid β protein fragment 25 35/product/Millipore
    Average 94 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    amyloid β protein fragment 25 35 - by Bioz Stars, 2020-05
    94/100 stars
      Buy from Supplier

    99
    Millipore anti flag epitope
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Anti Flag Epitope, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag epitope/product/Millipore
    Average 99 stars, based on 78 article reviews
    Price from $9.99 to $1999.99
    anti flag epitope - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    88
    Covance ha11
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Ha11, supplied by Covance, used in various techniques. Bioz Stars score: 88/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ha11/product/Covance
    Average 88 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    ha11 - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    99
    Millipore horseradish peroxidase
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Horseradish Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase/product/Millipore
    Average 99 stars, based on 13262 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    90
    Biomol GmbH fk2
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Fk2, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 90/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fk2/product/Biomol GmbH
    Average 90 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
    fk2 - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    88
    Millipore m5
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    M5, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m5/product/Millipore
    Average 88 stars, based on 51 article reviews
    Price from $9.99 to $1999.99
    m5 - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    89
    Covance anti ha epitope
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Anti Ha Epitope, supplied by Covance, used in various techniques. Bioz Stars score: 89/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ha epitope/product/Covance
    Average 89 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    anti ha epitope - by Bioz Stars, 2020-05
    89/100 stars
      Buy from Supplier

    92
    Millipore goat
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    Goat, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat/product/Millipore
    Average 92 stars, based on 1178 article reviews
    Price from $9.99 to $1999.99
    goat - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    98
    Millipore 6 hydroxdopamine
    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor <t>Z‐VAD‐FMK</t> (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.
    6 Hydroxdopamine, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 hydroxdopamine/product/Millipore
    Average 98 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    6 hydroxdopamine - by Bioz Stars, 2020-05
    98/100 stars
      Buy from Supplier

    92
    Biomol GmbH anti ubiquitin
    RNF170 possesses <t>ubiquitin</t> ligase activity. RNF170 FLAG ( lanes 1–4 ) and *RNF170 FLAG ( lane 5 ), immunopurified from transfected HeLa cells, were incubated with E1 ( UBE1 ), E2 ( UbcH5b ) and HA-ubiquitin as indicated for 30 min at 30 °C. Samples
    Anti Ubiquitin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ubiquitin/product/Biomol GmbH
    Average 92 stars, based on 136 article reviews
    Price from $9.99 to $1999.99
    anti ubiquitin - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    Image Search Results


    Inactivation of p97 impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    doi: 10.3389/fncel.2015.00016

    Figure Lengend Snippet: Inactivation of p97 impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p

    Article Snippet: For pharmacological inhibition of p97 function, SH-SY5Y cells were treated with the p97-inhibitor DBeQ (Sigma, SML0031) at 2.5 μM for 20 h before further analysis (Chou et al., ).

    Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Staining, Flow Cytometry, Cytometry, Fluorescence

    Inactivation of p97 increased mitochondrial fragmentation under stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 16 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for 6 h. Cells were fixed and stained using anti-cytochrome c antibody and Alexa546-conjugated secondary antibody. Shown are representative pictures from three independent experiments. (B) Fragmentation of the mitochondrial network in cells from (A) was quantified by visual examination. Shown is the average of three independent experiments. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Statistical significance is marked with n.s. for not significant, *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    doi: 10.3389/fncel.2015.00016

    Figure Lengend Snippet: Inactivation of p97 increased mitochondrial fragmentation under stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 16 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for 6 h. Cells were fixed and stained using anti-cytochrome c antibody and Alexa546-conjugated secondary antibody. Shown are representative pictures from three independent experiments. (B) Fragmentation of the mitochondrial network in cells from (A) was quantified by visual examination. Shown is the average of three independent experiments. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Statistical significance is marked with n.s. for not significant, *** p

    Article Snippet: For pharmacological inhibition of p97 function, SH-SY5Y cells were treated with the p97-inhibitor DBeQ (Sigma, SML0031) at 2.5 μM for 20 h before further analysis (Chou et al., ).

    Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Staining

    p97 is involved in mitochondrial maintenance at damage levels below the thresholds for mitophagy and cell death . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were transfected with an expression construct for YFP-tagged Parkin, induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were fixed, stained for the mitochondrial marker cytochrome c (cyt. c) and Parkin translocation from the cytosol to mitochondria was visually analyzed using fluorescence microscopy. Shown are representative images of three independent experiments. (B) Shown is a quantification of Parkin translocation from the cytosol to mitochondria from (A) . (C) Cells treated as in (A) were stained with the cell impermeable dye 4′,6-diamidino-2-phenylindole (DAPI) and the percentage of dead cells was determined by flow cytometry. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Statistical significance is marked with *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    doi: 10.3389/fncel.2015.00016

    Figure Lengend Snippet: p97 is involved in mitochondrial maintenance at damage levels below the thresholds for mitophagy and cell death . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were transfected with an expression construct for YFP-tagged Parkin, induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were fixed, stained for the mitochondrial marker cytochrome c (cyt. c) and Parkin translocation from the cytosol to mitochondria was visually analyzed using fluorescence microscopy. Shown are representative images of three independent experiments. (B) Shown is a quantification of Parkin translocation from the cytosol to mitochondria from (A) . (C) Cells treated as in (A) were stained with the cell impermeable dye 4′,6-diamidino-2-phenylindole (DAPI) and the percentage of dead cells was determined by flow cytometry. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Statistical significance is marked with *** p

    Article Snippet: For pharmacological inhibition of p97 function, SH-SY5Y cells were treated with the p97-inhibitor DBeQ (Sigma, SML0031) at 2.5 μM for 20 h before further analysis (Chou et al., ).

    Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Transfection, Construct, Staining, Marker, Translocation Assay, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Clearance of oxidatively-damaged mitochondrial proteins is impaired following inactivation of p97 . SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Mitochondria were isolated using anti-TOMM22 magnetic beads resulting in highly purified mitochondria. Protein carbonylation as measure for oxidative damage was determined by infrared laser-based quantitative western blotting following derivatization with 2,4-Dinitrophenylhydrazine (DNPH) and detection using anti-DNP antibodies. Shown is the average of three independent experiments. Please see Supplementary Figure S1 for representative western blot images. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Comparisons shown are tetracycline-induced, untreated cells vs. tetracycline-induced, treated cells. Statistical significance is marked with n.s. for not significant, *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    doi: 10.3389/fncel.2015.00016

    Figure Lengend Snippet: Clearance of oxidatively-damaged mitochondrial proteins is impaired following inactivation of p97 . SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Mitochondria were isolated using anti-TOMM22 magnetic beads resulting in highly purified mitochondria. Protein carbonylation as measure for oxidative damage was determined by infrared laser-based quantitative western blotting following derivatization with 2,4-Dinitrophenylhydrazine (DNPH) and detection using anti-DNP antibodies. Shown is the average of three independent experiments. Please see Supplementary Figure S1 for representative western blot images. Statistical analysis was performed using pair-wise t -tests with p -value adjustment according to Holm. Comparisons shown are tetracycline-induced, untreated cells vs. tetracycline-induced, treated cells. Statistical significance is marked with n.s. for not significant, *** p

    Article Snippet: For pharmacological inhibition of p97 function, SH-SY5Y cells were treated with the p97-inhibitor DBeQ (Sigma, SML0031) at 2.5 μM for 20 h before further analysis (Chou et al., ).

    Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Isolation, Magnetic Beads, Purification, Western Blot

    Inactivation of p97 impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    doi: 10.3389/fncel.2015.00016

    Figure Lengend Snippet: Inactivation of p97 impairs mitochondrial function during neurotoxic stress . (A) SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ under control of the Tet-On promoter were induced with tetracycline for 2 h or left uninduced and treated with 5 μM rotenone, 75 μM 6 OHDA, or 50 μM Aβ for an additional 6 h. Cells were stained with the mitochondrial membrane sensitive dye TMRE and analyzed by flow cytometry. (B) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial membrane potential was measured by flow cytometric analysis of TMRE fluorescence. (C) Cells treated as in (A) were stained with the ROS-sensitive dye MitoSox and mitochondrial ROS generation was measured using flow cytometry. (D) SH-SY5Y cells were treated with the p97 inhibitor DBeQ and mitochondrial ROS generation was measured by flow cytometric analysis of MitoSox fluorescence. Statistical analysis was performed using pair-wise t -tests with Holm p -value adjustment for (A,C) , and a general linear model (SPSS) for (B,D) . Statistical significance is marked with n.s. for not significant, # p

    Article Snippet: For pharmacological inhibition of p97 function, SH-SY5Y cells were treated with the p97-inhibitor DBeQ (Sigma, SML0031) at 2.5 μM for 20 h before further analysis (Chou et al., ).

    Techniques: Stable Transfection, Expressing, Dominant Negative Mutation, Staining, Flow Cytometry, Cytometry, Fluorescence

    MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of NMS‐873, MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: MITOL ∆C8 ubiquitylates expanded peroxisomes following CCCP treatment HeLa cells expressing HA‐Parkin and MITOL∆C8 (A) or MITOL∆C8 lacking E3 activity (B) were treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag and anti‐ubiquitin antibodies. Expanded peroxisomes were ubiquitylated upon E3 activity of MITOL. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. The schematic model of PINK1/Parkin‐mediated MITOL redistribution. MITOL is targeted to mitochondria with sustained membrane potential via Tom70. In response to mitophagy stimuli, Parkin‐catalyzed ubiquitylation of MITOL causes its extraction from damaged mitochondria in a p97/VCP‐dependent manner, and then, MITOL translocates to peroxisomes via Pex3/Pex16 pathway. As ubiquitylation of MITOL is rarely observed in the absence of NMS‐873, MITOL inserted into the peroxisome membrane is shown here in the non‐ubiquitylated form.

    Article Snippet: To inhibit proteasome activity, 10 μM MG132 (SIGMA) was used, and to inhibit p97/VCP activity, 10 μM NMS‐873 (SIGMA) was used.

    Techniques: Expressing, Activity Assay, Immunocytochemistry

    MITOL is extracted from mitochondria in a p97/ VCP ATP ase‐dependent manner MITOL did not merge with Hsp60 in control siRNA‐treated cells, whereas most MITOL co‐localized with Hsp60 in p97 / VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag‐MITOL and HA‐Parkin were transfected with control or p97 / VCP siRNA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag, anti‐catalase, and anti‐Hsp60 antibodies. Scale bars, 10 μm. HeLa cell lysates treated with control or p97 / VCP siRNA were immunoblotted with anti‐p97/VCP and anti‐tubulin antibodies. Overexpression of a p97/VCP ATP hydrolysis‐defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild‐type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA and Flag‐p97/VCP wild‐type or p97QQ, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐catalase antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. NMS‐873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP treatment. HeLa cells stably expressing HA‐Parkin were transfected with 3Flag‐MITOL, treated with 15 μM CCCP in the presence or absence of 10 μM NMS‐873 for 3 h, and then subjected to immunostaining with anti‐Flag, anti‐catalase, and anti‐HA antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Arrowheads in (C) and (D) indicate representative examples of MITOL–peroxisome co‐localization that was only observed in the presence of a functional VCP. Correlation statistics for the localization of 3Flag‐MITOL and catalase in the presence of NMS‐873. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: MITOL is extracted from mitochondria in a p97/ VCP ATP ase‐dependent manner MITOL did not merge with Hsp60 in control siRNA‐treated cells, whereas most MITOL co‐localized with Hsp60 in p97 / VCP knockdown cells in response to mitophagy stimuli. HeLa cells stably expressing 3Flag‐MITOL and HA‐Parkin were transfected with control or p97 / VCP siRNA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐Flag, anti‐catalase, and anti‐Hsp60 antibodies. Scale bars, 10 μm. HeLa cell lysates treated with control or p97 / VCP siRNA were immunoblotted with anti‐p97/VCP and anti‐tubulin antibodies. Overexpression of a p97/VCP ATP hydrolysis‐defective mutant, E305Q/E578Q (p97QQ), blocked MITOL redistribution from mitochondria to peroxisomes, while overexpression of wild‐type p97/VCP had no effect on MITOL redistribution. HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA and Flag‐p97/VCP wild‐type or p97QQ, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐catalase antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. NMS‐873, a specific inhibitor of p97/VCP, prevented MITOL translocation following CCCP treatment. HeLa cells stably expressing HA‐Parkin were transfected with 3Flag‐MITOL, treated with 15 μM CCCP in the presence or absence of 10 μM NMS‐873 for 3 h, and then subjected to immunostaining with anti‐Flag, anti‐catalase, and anti‐HA antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Arrowheads in (C) and (D) indicate representative examples of MITOL–peroxisome co‐localization that was only observed in the presence of a functional VCP. Correlation statistics for the localization of 3Flag‐MITOL and catalase in the presence of NMS‐873. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test.

    Article Snippet: To inhibit proteasome activity, 10 μM MG132 (SIGMA) was used, and to inhibit p97/VCP activity, 10 μM NMS‐873 (SIGMA) was used.

    Techniques: Stable Transfection, Expressing, Transfection, Immunocytochemistry, Over Expression, Mutagenesis, Translocation Assay, Immunostaining, Functional Assay, Software, One-tailed Test

    Endogenous MITOL translocates to peroxisomes To generate MITOL‐3Flag knock‐in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9‐based gene editing. Insertion of the 3Flag‐tag was verified by immunoblotting with an anti‐Flag antibody. Asterisk indicates a cross‐reacting band. The MITOL subcellular localization was observed in MITOL‐3Flag KI HCT116 cells stably expressing HA‐Parkin with anti‐Flag, anti‐catalase, and anti‐Hsp60 antibodies. Endogenous MITOL (detectable with an anti‐Flag antibody) overlapped with Hsp60 under steady‐state conditions, whereas 3 h of valinomycin (10 μM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm. Correlation statistics for the localization of endogenous MITOL‐3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines show the medians, box limits indicate the 25 th and 75 th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and the X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS‐873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 μm. Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS‐873. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines show the medians, box limits indicate the 25 th and 75 th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and the X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL‐3Flag KI HCT116 cells stably expressing HA‐Parkin were treated with valinomycin for 3 h, the collected cell lysates were immunoprecipitated with anti‐Flag magnetic beads. The immunoprecipitates were blotted using anti‐Flag and anti‐ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL‐3Flag.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: Endogenous MITOL translocates to peroxisomes To generate MITOL‐3Flag knock‐in (KI) HCT116 cell lines, 3xFlag gene cassettes were inserted upstream of the MITOL stop codon using CRISPR/Cas9‐based gene editing. Insertion of the 3Flag‐tag was verified by immunoblotting with an anti‐Flag antibody. Asterisk indicates a cross‐reacting band. The MITOL subcellular localization was observed in MITOL‐3Flag KI HCT116 cells stably expressing HA‐Parkin with anti‐Flag, anti‐catalase, and anti‐Hsp60 antibodies. Endogenous MITOL (detectable with an anti‐Flag antibody) overlapped with Hsp60 under steady‐state conditions, whereas 3 h of valinomycin (10 μM) treatment induced translocation of endogenous MITOL from mitochondria to peroxisomes. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm. Correlation statistics for the localization of endogenous MITOL‐3Flag with catalase. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines show the medians, box limits indicate the 25 th and 75 th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and the X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. The extraction of endogenous MITOL from depolarized mitochondria is blocked by the p97/VCP inhibitor NMS‐873. Higher magnification images of the boxed regions are shown in the lower panel. Scale bars, 10 μm. Correlation statistics for the localization of endogenous MITOL and PMP70 in the presence of NMS‐873. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines show the medians, box limits indicate the 25 th and 75 th percentiles as determined by the R software package, and whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and the X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. Endogenous MITOL is ubiquitylated following valinomycin treatment. After MITOL‐3Flag KI HCT116 cells stably expressing HA‐Parkin were treated with valinomycin for 3 h, the collected cell lysates were immunoprecipitated with anti‐Flag magnetic beads. The immunoprecipitates were blotted using anti‐Flag and anti‐ubiquitin antibodies. Red bars indicate ubiquitylation; the black arrowhead indicates MITOL‐3Flag.

    Article Snippet: To inhibit proteasome activity, 10 μM MG132 (SIGMA) was used, and to inhibit p97/VCP activity, 10 μM NMS‐873 (SIGMA) was used.

    Techniques: Knock-In, CRISPR, Stable Transfection, Expressing, Translocation Assay, Software, One-tailed Test, Immunoprecipitation, Magnetic Beads

    MITOL K268, K40, and K54 are ubiquitylated in response to mitophagy An increased molecular weight shift of MITOL based on ubiquitylation was observed following addition of the p97/VCP specific inhibitor NMS‐873. After 3 h of 15 μM CCCP treatment with or without 10 μM NMS‐873, cell lysates from HeLa cells stably expressing HA‐Parkin and 3Flag‐MITOL were analyzed by immunoblotting with an anti‐Flag antibody. Red bar indicates ubiquitylation. Mass spectrometric (MS) analysis for identification of MITOL ubiquitylation sites. PEX19 −/− HCT116 cells stably expressing HA‐Parkin and 3Flag‐MITOL after 3 h of valinomycin treatment were immunoprecipitated with anti‐Flag magnetic beads, and then subjected to LC‐MS/MS analysis. Ubiquitylation of K268, K40, and K54 was revealed by the MS/MS spectra of m / z 724.35694, 562.28265, and 358.84908 ions in tryptic peptides, respectively. The abundances of K268, K40, and K54 peptides with di‐glycine modification in valinomycin‐treated samples were compared with those determined in control (untreated) samples. MITOL K268 underwent significant ubiquitylation in response to mitophagy. Error bars represent the mean ± s.e.m. in three biological replicates. Statistical significance was calculated using a one‐tailed Student's t ‐test. Multiple sequence alignment of the MITOL C‐terminal region containing K268 from various organisms. K268 (red font) is well conserved. Gray shading indicates the fourth transmembrane segment.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: MITOL K268, K40, and K54 are ubiquitylated in response to mitophagy An increased molecular weight shift of MITOL based on ubiquitylation was observed following addition of the p97/VCP specific inhibitor NMS‐873. After 3 h of 15 μM CCCP treatment with or without 10 μM NMS‐873, cell lysates from HeLa cells stably expressing HA‐Parkin and 3Flag‐MITOL were analyzed by immunoblotting with an anti‐Flag antibody. Red bar indicates ubiquitylation. Mass spectrometric (MS) analysis for identification of MITOL ubiquitylation sites. PEX19 −/− HCT116 cells stably expressing HA‐Parkin and 3Flag‐MITOL after 3 h of valinomycin treatment were immunoprecipitated with anti‐Flag magnetic beads, and then subjected to LC‐MS/MS analysis. Ubiquitylation of K268, K40, and K54 was revealed by the MS/MS spectra of m / z 724.35694, 562.28265, and 358.84908 ions in tryptic peptides, respectively. The abundances of K268, K40, and K54 peptides with di‐glycine modification in valinomycin‐treated samples were compared with those determined in control (untreated) samples. MITOL K268 underwent significant ubiquitylation in response to mitophagy. Error bars represent the mean ± s.e.m. in three biological replicates. Statistical significance was calculated using a one‐tailed Student's t ‐test. Multiple sequence alignment of the MITOL C‐terminal region containing K268 from various organisms. K268 (red font) is well conserved. Gray shading indicates the fourth transmembrane segment.

    Article Snippet: To inhibit proteasome activity, 10 μM MG132 (SIGMA) was used, and to inhibit p97/VCP activity, 10 μM NMS‐873 (SIGMA) was used.

    Techniques: Molecular Weight, Stable Transfection, Expressing, Mass Spectrometry, Immunoprecipitation, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Modification, One-tailed Test, Sequencing

    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z‐VAD‐FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z‐VAD‐FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.

    Article Snippet: To inhibit proteasome activity, 10 μM MG132 (SIGMA) was used, and to inhibit p97/VCP activity, 10 μM NMS‐873 (SIGMA) was used.

    Techniques: Stable Transfection, Expressing, Transfection, Immunocytochemistry, Software, One-tailed Test, Plasmid Preparation, Fractionation

    Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z‐VAD‐FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.

    Journal: EMBO Reports

    Article Title: Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

    doi: 10.15252/embr.201947728

    Figure Lengend Snippet: Parkin is required for MITOL redistribution to peroxisomes MITOL‐HA did not move to peroxisomes, but was rather retained on mitochondria even after CCCP treatment in HeLa cells lacking endogenous Parkin. Wild‐type HeLa cells or HeLa cells stably expressing GFP‐Parkin were transfected with MITOL‐HA, treated with 15 μM CCCP for 3 h, and then subjected to immunocytochemistry with anti‐HA and anti‐Tom20 antibodies. Higher magnification images of the boxed regions are shown in the small panel. Scale bars, 10 μm. Correlation statistics for the localization of MITOL‐HA and Tom20 in the absence or presence of GFP‐Parkin. Dots indicate individual Pearson correlation coefficient data points. In the box‐plots, the center lines indicate the medians, the box limits indicate the 25 th and 75 th percentiles as determined in the R software package, and the whiskers extend 1.5 times the interquartile range from the 25 th and 75 th percentiles. Means and the number of samples are shown on the box and X ‐axis, respectively. Statistical significance was calculated using a one‐tailed Welch's t ‐test. MITOL was not degraded following mitochondrial depolarization. HeLa cells stably expressing 3Flag‐MITOL were transfected with a GFP‐Parkin plasmid or the pEGFP‐C1 vector, treated with 15 μM CCCP ± 10 μM MG132 for 3 h, and then immunoblotted with anti‐Flag, anti‐ubiquitin, and anti‐tubulin antibodies. Black arrowheads indicate 3Flag‐MITOL in the upper panel and mono‐ubiquitin in the middle panel, respectively. The total cellular amount of MITOL was not dramatically reduced following extended valinomycin treatment. Immunoblotting combined with fractionation analysis showed that mitofusin2 (MFN2) underwent rapid degradation within 3 h of valinomycin treatment, in particular in the 3,000 g pellet (mitochondria‐rich fractions). Cytochrome c oxidase subunit 2 (MTCO2, inner mitochondrial protein) was significantly reduced at 24 h 10 μM valinomycin treatment. In contrast to those two proteins, MITOL degradation was minimal. Note that the chemical apoptosis inhibitor Z‐VAD‐FMK (10 μM) was added to cells along with valinomycin to prevent cell death. Quantification of 3Flag‐MITOL, MFN2, and MTCO2 protein levels in the PNS and 3,000 g pellet fraction following 10 μM valinomycin + Z‐VAD‐FMK treatment at the indicated times. Data represent the mean fold change ± s.e.m. relative to untreated samples in three biological replicates. Pre‐existing MITOL on mitochondria moves to peroxisomes following CCCP treatment. Following doxycycline treatment for 3 h to induce MITOL expression, cells were washed with fresh medium to stop the synthesis of new MITOL. After treatment with or without CCCP for more than 3 h, cells were immunostained using anti‐Flag, anti‐Pex14 (peroxisomal membrane protein), and anti‐Hsp60 antibodies. Higher magnification images of the boxed regions are shown in the bottom panel. Scale bars, 10 μm.

    Article Snippet: To prevent SH‐SY5Y cell death in response to the valinomycin treatment, an apoptosis inhibitor, Z‐VAD‐FMK (Peptide Institute), was included in the incubation.

    Techniques: Stable Transfection, Expressing, Transfection, Immunocytochemistry, Software, One-tailed Test, Plasmid Preparation, Fractionation

    RNF170 possesses ubiquitin ligase activity. RNF170 FLAG ( lanes 1–4 ) and *RNF170 FLAG ( lane 5 ), immunopurified from transfected HeLa cells, were incubated with E1 ( UBE1 ), E2 ( UbcH5b ) and HA-ubiquitin as indicated for 30 min at 30 °C. Samples

    Journal: The Journal of Biological Chemistry

    Article Title: RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation *

    doi: 10.1074/jbc.M111.251983

    Figure Lengend Snippet: RNF170 possesses ubiquitin ligase activity. RNF170 FLAG ( lanes 1–4 ) and *RNF170 FLAG ( lane 5 ), immunopurified from transfected HeLa cells, were incubated with E1 ( UBE1 ), E2 ( UbcH5b ) and HA-ubiquitin as indicated for 30 min at 30 °C. Samples

    Article Snippet: Already available antibodies used were rabbit polyclonal anti-IP3 R1 , anti-erlin2 , anti-FLAG epitope , anti-α-transaldolase (a kind gift from Dr. A. Perl, State University of New York Upstate Medical University, Syracuse, NY), mouse monoclonal anti-p97 (Research Diagnostics, Inc.), anti-ubiquitin clone FK2 (BioMol International), anti-FLAG epitope clone M2 or M5 (Sigma), anti-HA epitope clone HA11 (Covance), and horseradish peroxidase- and fluorophore-conjugated secondary antibodies raised in goat (Sigma).

    Techniques: Activity Assay, Transfection, Incubation