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    Structured Review

    Cell Signaling Technology Inc wfs1
    ( a ) Affinity purification followed by tandem mass spectrometry analysis was conducted to identify mtHtt-binding proteins on mitochondria in HdhQ7 and HdhQ111 striatal cells. The molecular and cellular function of the exclusive mtHtt interactors on mitochondria of HdhQ111 cells are shown. VCP was the leading candidate for an mtHtt-binding protein . ( b ) Mitochondrial and ER fractions were isolated from HdhQ7 and HdhQ111 cells. Protein levels of VCP were analysed by western blotting (WB). VDAC and <t>WFS1</t> were used as loading controls for mitochondria and ER. Data are mean±s.e.m. of at least three independent experiments. ( c ) Control siRNA (Con) and Htt siRNA (siHTT) were transfected in HdhQ7 and HdhQ111 cells for 3 days, respectively. VCP levels were determined in mitochondrial fractions by WB analysis. VDAC was a loading control. Data are mean±s.e.m. of at least three independent experiments (One-way ANOVA with Holm-Sidak post hoc test). ( d ) HdhQ7 and HdhQ111 cells were stained with anti-Tom20 (green, a mitochondrial marker) and anti-VCP (red) antibodies. VCP/Tom20 co-localization was examined using confocal microscopy. Scale bar: 10 μm. Pearson's co-efficiency was calculated. At least 100 cells per group were counted. Data are mean±s.e.m. of three independent experiments. ( e ) Immunogold electron microscopy analysis of VCP on mitochondria was conducted. Scale bar: 100 nm. The number of gold particles labelling VCP was quantitated and shown as mean±s.e.m. A total of 30 mitochondria from each group were counted. * P <0.01 versus HdhQ7 cells. ( f ) Mitochondria were isolated from the striata of either HD transgenic mice R6/2 (9-week-old) or YAC128 (6-month-old). n =6 mice/group. VCP levels were determined by WB (loading control: VDAC). ( g ) Paraffin-embedded sections (5 μm thick) of the caudate nucleus from three HD patients (ID: 2982, 2983 and 5413) and three normal subjects (ID: 623, 624 and 1533) were immunostained with anti-VCP (red) and anti-Tom20 (green) antibodies. Localization of VCP on mitochondria was examined using confocal microscopy. Pearson's co-efficiency was calculated. Scale bar: 10 μm. Data are mean±s.e.m. ( b , d – g ) Paired Student's t -test.
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    1) Product Images from "VCP recruitment to mitochondria causes mitophagy impairment and neurodegeneration in models of Huntington's disease"

    Article Title: VCP recruitment to mitochondria causes mitophagy impairment and neurodegeneration in models of Huntington's disease

    Journal: Nature Communications

    doi: 10.1038/ncomms12646

    ( a ) Affinity purification followed by tandem mass spectrometry analysis was conducted to identify mtHtt-binding proteins on mitochondria in HdhQ7 and HdhQ111 striatal cells. The molecular and cellular function of the exclusive mtHtt interactors on mitochondria of HdhQ111 cells are shown. VCP was the leading candidate for an mtHtt-binding protein . ( b ) Mitochondrial and ER fractions were isolated from HdhQ7 and HdhQ111 cells. Protein levels of VCP were analysed by western blotting (WB). VDAC and WFS1 were used as loading controls for mitochondria and ER. Data are mean±s.e.m. of at least three independent experiments. ( c ) Control siRNA (Con) and Htt siRNA (siHTT) were transfected in HdhQ7 and HdhQ111 cells for 3 days, respectively. VCP levels were determined in mitochondrial fractions by WB analysis. VDAC was a loading control. Data are mean±s.e.m. of at least three independent experiments (One-way ANOVA with Holm-Sidak post hoc test). ( d ) HdhQ7 and HdhQ111 cells were stained with anti-Tom20 (green, a mitochondrial marker) and anti-VCP (red) antibodies. VCP/Tom20 co-localization was examined using confocal microscopy. Scale bar: 10 μm. Pearson's co-efficiency was calculated. At least 100 cells per group were counted. Data are mean±s.e.m. of three independent experiments. ( e ) Immunogold electron microscopy analysis of VCP on mitochondria was conducted. Scale bar: 100 nm. The number of gold particles labelling VCP was quantitated and shown as mean±s.e.m. A total of 30 mitochondria from each group were counted. * P <0.01 versus HdhQ7 cells. ( f ) Mitochondria were isolated from the striata of either HD transgenic mice R6/2 (9-week-old) or YAC128 (6-month-old). n =6 mice/group. VCP levels were determined by WB (loading control: VDAC). ( g ) Paraffin-embedded sections (5 μm thick) of the caudate nucleus from three HD patients (ID: 2982, 2983 and 5413) and three normal subjects (ID: 623, 624 and 1533) were immunostained with anti-VCP (red) and anti-Tom20 (green) antibodies. Localization of VCP on mitochondria was examined using confocal microscopy. Pearson's co-efficiency was calculated. Scale bar: 10 μm. Data are mean±s.e.m. ( b , d – g ) Paired Student's t -test.
    Figure Legend Snippet: ( a ) Affinity purification followed by tandem mass spectrometry analysis was conducted to identify mtHtt-binding proteins on mitochondria in HdhQ7 and HdhQ111 striatal cells. The molecular and cellular function of the exclusive mtHtt interactors on mitochondria of HdhQ111 cells are shown. VCP was the leading candidate for an mtHtt-binding protein . ( b ) Mitochondrial and ER fractions were isolated from HdhQ7 and HdhQ111 cells. Protein levels of VCP were analysed by western blotting (WB). VDAC and WFS1 were used as loading controls for mitochondria and ER. Data are mean±s.e.m. of at least three independent experiments. ( c ) Control siRNA (Con) and Htt siRNA (siHTT) were transfected in HdhQ7 and HdhQ111 cells for 3 days, respectively. VCP levels were determined in mitochondrial fractions by WB analysis. VDAC was a loading control. Data are mean±s.e.m. of at least three independent experiments (One-way ANOVA with Holm-Sidak post hoc test). ( d ) HdhQ7 and HdhQ111 cells were stained with anti-Tom20 (green, a mitochondrial marker) and anti-VCP (red) antibodies. VCP/Tom20 co-localization was examined using confocal microscopy. Scale bar: 10 μm. Pearson's co-efficiency was calculated. At least 100 cells per group were counted. Data are mean±s.e.m. of three independent experiments. ( e ) Immunogold electron microscopy analysis of VCP on mitochondria was conducted. Scale bar: 100 nm. The number of gold particles labelling VCP was quantitated and shown as mean±s.e.m. A total of 30 mitochondria from each group were counted. * P <0.01 versus HdhQ7 cells. ( f ) Mitochondria were isolated from the striata of either HD transgenic mice R6/2 (9-week-old) or YAC128 (6-month-old). n =6 mice/group. VCP levels were determined by WB (loading control: VDAC). ( g ) Paraffin-embedded sections (5 μm thick) of the caudate nucleus from three HD patients (ID: 2982, 2983 and 5413) and three normal subjects (ID: 623, 624 and 1533) were immunostained with anti-VCP (red) and anti-Tom20 (green) antibodies. Localization of VCP on mitochondria was examined using confocal microscopy. Pearson's co-efficiency was calculated. Scale bar: 10 μm. Data are mean±s.e.m. ( b , d – g ) Paired Student's t -test.

    Techniques Used: Affinity Purification, Mass Spectrometry, Binding Assay, Cell Function Assay, Isolation, Western Blot, Transfection, Staining, Marker, Confocal Microscopy, Electron Microscopy, Transgenic Assay

    ( a ) Mitochondrial, ER, and cytosolic fractions (Ct) of HdhQ7 and HdhQ111 mouse striatal cells were subjected to immunoprecipitation (IP) with anti-VCP antibody, and immunoprecipitates were analysed by WB with anti-VCP and anti-MAB2166 antibody (recognizes both wt and mtHtt, left panel) or anti-1C2 antibody (recognizes mtHtt, right panel). Note that polyQ protein above 250 kDa is shown in the right panel. Representative blots from three independent experiments are shown. ( b ) Mitochondrial, ER, and cytosolic fractions were isolated from striata of YAC128 and wild-type mice at the age of 6 months. IP with anti-VCP antibody followed by anti-1C2 antibody or anti-EM48 antibody was performed. The right panel indicates the purity of ER and mitochondrial fractions isolated from YAC128 mouse striata. WFS1 and VDAC were used to label ER and mitochondria, respectively. n =4 mice/group. ( c ) Mitochondrial, ER and cytosolic fractions were isolated from fibroblasts of HD patients (HD1 carries 70 CAG repeats and HD2 carries 60 CAG repeats, respectively) and normal subjects. IP with anti-VCP antibody followed by WB with anti-1C2 antibody was conducted. Representative blots from two independent experiments are shown. ( d ) Total cortical protein lysates from postmortem brain tissues of 4 normal subjects and 4 HD patients were subjected to IP with anti-VCP antibody followed by WB with anti-1C2 antibody. Arrows indicate mtHtt recognized by 1C2 antibody which does not detect wt Htt. Note: mtHtt protein around 82 kDa recognized by 1C2 antibody has been shown to be abundant in cortical tissues of HD mice and HD patients . The identity numbers of the HD patients (HD) and normal subjects (Nor) were listed on the bottom. HD patients (5348 and 5263) exhibited extensive neuronal loss and severe brain atrophy, and HD patients (5298 and 5496) showed moderate neuronal loss and brain atrophy. The information of normal subjects and HD patients was summarized in . Normal subjects had no history of HD or other neurological diseases.
    Figure Legend Snippet: ( a ) Mitochondrial, ER, and cytosolic fractions (Ct) of HdhQ7 and HdhQ111 mouse striatal cells were subjected to immunoprecipitation (IP) with anti-VCP antibody, and immunoprecipitates were analysed by WB with anti-VCP and anti-MAB2166 antibody (recognizes both wt and mtHtt, left panel) or anti-1C2 antibody (recognizes mtHtt, right panel). Note that polyQ protein above 250 kDa is shown in the right panel. Representative blots from three independent experiments are shown. ( b ) Mitochondrial, ER, and cytosolic fractions were isolated from striata of YAC128 and wild-type mice at the age of 6 months. IP with anti-VCP antibody followed by anti-1C2 antibody or anti-EM48 antibody was performed. The right panel indicates the purity of ER and mitochondrial fractions isolated from YAC128 mouse striata. WFS1 and VDAC were used to label ER and mitochondria, respectively. n =4 mice/group. ( c ) Mitochondrial, ER and cytosolic fractions were isolated from fibroblasts of HD patients (HD1 carries 70 CAG repeats and HD2 carries 60 CAG repeats, respectively) and normal subjects. IP with anti-VCP antibody followed by WB with anti-1C2 antibody was conducted. Representative blots from two independent experiments are shown. ( d ) Total cortical protein lysates from postmortem brain tissues of 4 normal subjects and 4 HD patients were subjected to IP with anti-VCP antibody followed by WB with anti-1C2 antibody. Arrows indicate mtHtt recognized by 1C2 antibody which does not detect wt Htt. Note: mtHtt protein around 82 kDa recognized by 1C2 antibody has been shown to be abundant in cortical tissues of HD mice and HD patients . The identity numbers of the HD patients (HD) and normal subjects (Nor) were listed on the bottom. HD patients (5348 and 5263) exhibited extensive neuronal loss and severe brain atrophy, and HD patients (5298 and 5496) showed moderate neuronal loss and brain atrophy. The information of normal subjects and HD patients was summarized in . Normal subjects had no history of HD or other neurological diseases.

    Techniques Used: Immunoprecipitation, Isolation