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rabbit polyclonal anti pink1 (Novus Biologicals)

Name: rabbit polyclonal anti pink1
Brand: Novus Biologicals
Rating: 96 (279 reviews)

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Novus Biologicals rabbit polyclonal anti pink1

(A) H1HeLa cells were transfected with the scramble and Parkin siRNAs for 48 h. The cells were then infected with EV-D68 for 5 h and viral titers were measured by a plaque assay. (B) H1HeLa cells were transfected with siRNAs against the mitophagy receptor, BCL2L13 for 48 h. The cells were then infected as in A for a plaque assay-based viral titer measurement. (C) A549 cells were transfected with scramble, Parkin, and <t>PINK1</t> siRNAs for 48 h. The cells were infected with EV-D68 for 5h and viral titers were determined by a plaque assay. (D) A549 cells were transfected with the indicated siRNAs for 48 h. The cells were then infected and tittered as in A. (E) A549 cells were transfected with the indicated mitophagy-associated siRNAs for 48 h, followed by viral infection and titer determination as in A. An MOI of 0.1 was used for all infections. Error bars indicate mean ± SEM of at least 2 independent repeats. Unpaired student’s t-test was used for the statistical analyses (**= p< 0.01; *= p< 0.05; ns=not significant.).

Rabbit Polyclonal Anti Pink1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 96/100, based on 279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 279 article reviews

rabbit polyclonal anti pink1 - by Bioz Stars, 2026-03

96/100 stars

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1) Product Images from "Mitophagosomes induced during EV-D68 infection promote viral nonlytic release"

Article Title: Mitophagosomes induced during EV-D68 infection promote viral nonlytic release

Journal: bioRxiv

doi: 10.1101/2024.12.05.627125

Figure Legend Snippet: (A) H1HeLa cells were transfected with the scramble and Parkin siRNAs for 48 h. The cells were then infected with EV-D68 for 5 h and viral titers were measured by a plaque assay. (B) H1HeLa cells were transfected with siRNAs against the mitophagy receptor, BCL2L13 for 48 h. The cells were then infected as in A for a plaque assay-based viral titer measurement. (C) A549 cells were transfected with scramble, Parkin, and PINK1 siRNAs for 48 h. The cells were infected with EV-D68 for 5h and viral titers were determined by a plaque assay. (D) A549 cells were transfected with the indicated siRNAs for 48 h. The cells were then infected and tittered as in A. (E) A549 cells were transfected with the indicated mitophagy-associated siRNAs for 48 h, followed by viral infection and titer determination as in A. An MOI of 0.1 was used for all infections. Error bars indicate mean ± SEM of at least 2 independent repeats. Unpaired student’s t-test was used for the statistical analyses (**= p< 0.01; *= p< 0.05; ns=not significant.).

Techniques Used: Transfection, Infection, Plaque Assay

Image Search Results

Journal: bioRxiv

Article Title: Mitophagosomes induced during EV-D68 infection promote viral nonlytic release

doi: 10.1101/2024.12.05.627125

Figure Lengend Snippet: (A) H1HeLa cells were transfected with the scramble and Parkin siRNAs for 48 h. The cells were then infected with EV-D68 for 5 h and viral titers were measured by a plaque assay. (B) H1HeLa cells were transfected with siRNAs against the mitophagy receptor, BCL2L13 for 48 h. The cells were then infected as in A for a plaque assay-based viral titer measurement. (C) A549 cells were transfected with scramble, Parkin, and PINK1 siRNAs for 48 h. The cells were infected with EV-D68 for 5h and viral titers were determined by a plaque assay. (D) A549 cells were transfected with the indicated siRNAs for 48 h. The cells were then infected and tittered as in A. (E) A549 cells were transfected with the indicated mitophagy-associated siRNAs for 48 h, followed by viral infection and titer determination as in A. An MOI of 0.1 was used for all infections. Error bars indicate mean ± SEM of at least 2 independent repeats. Unpaired student’s t-test was used for the statistical analyses (**= p< 0.01; *= p< 0.05; ns=not significant.).

Article Snippet: Rabbit polyclonal-anti-Pink1 (Novus Biologicals, BC100-494).

Techniques: Transfection, Infection, Plaque Assay

PINK1 is a binding partner of FBW7β. A , HEK293 cells were transfected with plasmids encoding PINK1-Myc alone or together with FLAG-FBW7α, FLAG-FBW7β, or FLAG-FBW7γ for 24 h. Total cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. Tubulin served as a loading control. B , where indicated, SH-SY5Y cells were mock-transfected (−) or transfected with a plasmid encoding FLAG-FBW7β for 48 h, followed by treatment with 20 μM MG132 for 4 h before harvesting. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. Preimmune IgG was used as a negative control for immunoprecipitation. Tubulin served as a loading control. The asterisk indicates IgG heavy chains. The bands marked with a red arrowhead indicated the PINK1 bands. C , where indicated, HEK293 cells were cotransfected with plasmids encoding PINK1-Myc and one of the FLAG-FBW7α, FLAG-FBW7β, or FLAG-FBW7γ for 24 h. Representative confocal images of immunostained ectopic FBW7 isoforms and PINK1 are shown. The scale bar represents 5 μm. D , the representative confocal images of the colocalization of endogenous FBW7β ( red ) and PINK1 ( green ) in SH-SY5Y cells are shown. The scale bar represents 5 μm. E , PLAs were performed using primary antibodies against PINK1 and FBW7β. Representative PLA images ( red ) depicting the interaction between endogenous PINK1 and FBW7β are shown. The scale bar represents 5 μm. FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; PLA, proximity ligation assay.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: PINK1 is a binding partner of FBW7β. A , HEK293 cells were transfected with plasmids encoding PINK1-Myc alone or together with FLAG-FBW7α, FLAG-FBW7β, or FLAG-FBW7γ for 24 h. Total cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. Tubulin served as a loading control. B , where indicated, SH-SY5Y cells were mock-transfected (−) or transfected with a plasmid encoding FLAG-FBW7β for 48 h, followed by treatment with 20 μM MG132 for 4 h before harvesting. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. Preimmune IgG was used as a negative control for immunoprecipitation. Tubulin served as a loading control. The asterisk indicates IgG heavy chains. The bands marked with a red arrowhead indicated the PINK1 bands. C , where indicated, HEK293 cells were cotransfected with plasmids encoding PINK1-Myc and one of the FLAG-FBW7α, FLAG-FBW7β, or FLAG-FBW7γ for 24 h. Representative confocal images of immunostained ectopic FBW7 isoforms and PINK1 are shown. The scale bar represents 5 μm. D , the representative confocal images of the colocalization of endogenous FBW7β ( red ) and PINK1 ( green ) in SH-SY5Y cells are shown. The scale bar represents 5 μm. E , PLAs were performed using primary antibodies against PINK1 and FBW7β. Representative PLA images ( red ) depicting the interaction between endogenous PINK1 and FBW7β are shown. The scale bar represents 5 μm. FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; PLA, proximity ligation assay.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Negative Control, Proximity Ligation Assay

FBW7β negatively regulates the protein stability of PINK1. A , where indicated, HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with increasing doses of FLAG-FBW7β-WT. Whole-cell lysates were subsequently immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). B , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with either FLAG-FBW7β-WT or FLAG-FBW7β-ΔF. Whole-cell lysates were subsequently immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001). C , HEK293 cells were transfected for 48 h with plasmids encoding PINK1-Myc, nonspecific scrambled control siRNA, or FBW7β -siRNA alone or in combination. Cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗∗ p < 0.001). D , the FBW7 +/+ and FBW7 −/− HCT116 cells were mock-transfected or transfected with a plasmid encoding FLAG-FBW7β for 48 h. Whole-cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to HSP90 were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01). E , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with FLAG-FBW7β. Cells were then treated with 50 μg/ml cycloheximide (CHX) for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to HSP90 were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). F , HEK293 cells were transfected for 48 h with scrambled control siRNA, FBW7β -siRNA, or plasmid encoding Myc-tagged PINK1 alone or in combination. Cells were then treated with 50 μg/ml CHX for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). G , the FBW7 +/+ and FBW7 −/− HCT116 cells were treated with 50 μg/ml CHX for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to tubulin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). Tubulin, β-actin, and HSP90 were used as a control for equal protein loading. FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β negatively regulates the protein stability of PINK1. A , where indicated, HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with increasing doses of FLAG-FBW7β-WT. Whole-cell lysates were subsequently immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). B , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with either FLAG-FBW7β-WT or FLAG-FBW7β-ΔF. Whole-cell lysates were subsequently immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001). C , HEK293 cells were transfected for 48 h with plasmids encoding PINK1-Myc, nonspecific scrambled control siRNA, or FBW7β -siRNA alone or in combination. Cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗∗ p < 0.001). D , the FBW7 +/+ and FBW7 −/− HCT116 cells were mock-transfected or transfected with a plasmid encoding FLAG-FBW7β for 48 h. Whole-cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to HSP90 were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗∗ p < 0.01). E , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-Myc alone or together with FLAG-FBW7β. Cells were then treated with 50 μg/ml cycloheximide (CHX) for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to HSP90 were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). F , HEK293 cells were transfected for 48 h with scrambled control siRNA, FBW7β -siRNA, or plasmid encoding Myc-tagged PINK1 alone or in combination. Cells were then treated with 50 μg/ml CHX for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). G , the FBW7 +/+ and FBW7 −/− HCT116 cells were treated with 50 μg/ml CHX for the indicated times, and cell lysates were immunoblotted with the indicated antibodies. Relative PINK1 levels compared to tubulin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05). Tubulin, β-actin, and HSP90 were used as a control for equal protein loading. FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Transfection, Control, Plasmid Preparation

FBW7β promotes the degradation of PINK1 via the SCF complex–dependent proteasome pathway. A , HEK293 cells were transfected with plasmid encoding PINK1-Myc or FLAG-FBW7β alone or in combination for 24 h. Cells were then treated with vehicle, 20 μM MG132, or 2 μM epoxomicin for 6 h before harvesting. Cell lysates were subjected to immunoblotting with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant). B , after FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM MG132 for 6 h, cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05). C , where indicated, HEK293 cells were transfected with scrambled control siRNA or cullin-1 -siRNA for 48 h, and treated with 20 μM MG132 or 20 μM CCCP for additional 4 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to tubulin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). D , SH-SY5Y cells were treated with 1 μM MLN4924 for the indicated times and treated with 20 μM MG132 or 20 μM CCCP for additional 6 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). The CCCP treatment was employed to denote the size of endogenous full-length PINK1 band, while MG132 treatment was used to represent the size of endogenous cleaved PINK1 band. E , HEK293 cells were transfected with scrambled control siRNA, FBW7β -siRNA, or plasmid encoding PINK1-Myc alone or in combination for 48 h, and treated with vehicle or 2 μM MLN4924 for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to β-actin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant). Tubulin, β-actin, and HSP90 were used as a control for equal protein loading. CCCP, carbonyl cyanide 3-chlorophenylhydrazone; FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; SCF, Skp1-Cullin-1-F-box protein.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β promotes the degradation of PINK1 via the SCF complex–dependent proteasome pathway. A , HEK293 cells were transfected with plasmid encoding PINK1-Myc or FLAG-FBW7β alone or in combination for 24 h. Cells were then treated with vehicle, 20 μM MG132, or 2 μM epoxomicin for 6 h before harvesting. Cell lysates were subjected to immunoblotting with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant). B , after FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM MG132 for 6 h, cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05). C , where indicated, HEK293 cells were transfected with scrambled control siRNA or cullin-1 -siRNA for 48 h, and treated with 20 μM MG132 or 20 μM CCCP for additional 4 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to tubulin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). D , SH-SY5Y cells were treated with 1 μM MLN4924 for the indicated times and treated with 20 μM MG132 or 20 μM CCCP for additional 6 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to HSP90 were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). The CCCP treatment was employed to denote the size of endogenous full-length PINK1 band, while MG132 treatment was used to represent the size of endogenous cleaved PINK1 band. E , HEK293 cells were transfected with scrambled control siRNA, FBW7β -siRNA, or plasmid encoding PINK1-Myc alone or in combination for 48 h, and treated with vehicle or 2 μM MLN4924 for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative PINK1 levels compared to β-actin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant). Tubulin, β-actin, and HSP90 were used as a control for equal protein loading. CCCP, carbonyl cyanide 3-chlorophenylhydrazone; FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; SCF, Skp1-Cullin-1-F-box protein.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control

FBW7β promotes K48-linked polyubiquitination of PINK1. A , HEK293 cells were transfected with plasmids encoding PINK1-Myc, FLAG-FBW7β, or HA-tagged ubiquitin (HA-Ubi) alone or in combination for 24 h, and treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. B , HEK293 cells were transfected with plasmids encoding PINK1-V5-His, FLAG-FBW7β-WT, or FLAG-FBW7β-ΔF alone or in combination for 24 h, and incubated with 20 μM MG132 for an additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the indicated antibodies. C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with 10 μM MG132 for 6 h. Immunoprecipitation (IP) of cell lysates was performed using an anti-PINK1 antibody, followed by Western blotting with anti-ubiquitin (Ubi) or anti-PINK1 antibody. The red arrowheads indicated the PINK1 bands. D , HEK293 cells were transfected with plasmids encoding PINK1-Myc, FLAG-FBW7β, HA-Ubi-WT, or one of two HA-Ubi mutants where the Lys residues except for the numbered one (K48, K63) were replaced with Arg either alone or in combination for 24 h. Cells were then treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the specified antibodies. β-Actin and HSP90 were used as controls for equal protein loading. The asterisk indicates IgG heavy chains. FBW7, F-box and WD repeat domain–containing 7; HA, hemagglutinin; PINK1, PTEN-induced kinase 1.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β promotes K48-linked polyubiquitination of PINK1. A , HEK293 cells were transfected with plasmids encoding PINK1-Myc, FLAG-FBW7β, or HA-tagged ubiquitin (HA-Ubi) alone or in combination for 24 h, and treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the indicated antibodies. B , HEK293 cells were transfected with plasmids encoding PINK1-V5-His, FLAG-FBW7β-WT, or FLAG-FBW7β-ΔF alone or in combination for 24 h, and incubated with 20 μM MG132 for an additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the indicated antibodies. C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with 10 μM MG132 for 6 h. Immunoprecipitation (IP) of cell lysates was performed using an anti-PINK1 antibody, followed by Western blotting with anti-ubiquitin (Ubi) or anti-PINK1 antibody. The red arrowheads indicated the PINK1 bands. D , HEK293 cells were transfected with plasmids encoding PINK1-Myc, FLAG-FBW7β, HA-Ubi-WT, or one of two HA-Ubi mutants where the Lys residues except for the numbered one (K48, K63) were replaced with Arg either alone or in combination for 24 h. Cells were then treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-PINK1 antibody, followed by immunoblotting with the specified antibodies. β-Actin and HSP90 were used as controls for equal protein loading. The asterisk indicates IgG heavy chains. FBW7, F-box and WD repeat domain–containing 7; HA, hemagglutinin; PINK1, PTEN-induced kinase 1.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Incubation

FBW7β depletion regulates the levels of autophagy markers, increasing LC3 and decreasing p62, upon CCCP treatment. A , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). B , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗∗ p < 0.001). C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM CCCP for the indicated times. Cell lysates were immunoblotted with the specified antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). D , SH-SY5Y cells were treated with vehicle or 10 μM CCCP for 24 h and then treated with vehicle or 50 μM CQ for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗∗∗ p < 0.001). E , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 10 μM CCCP alone or in combination with 50 μM CQ for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001). FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CQ, chloroquine.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β depletion regulates the levels of autophagy markers, increasing LC3 and decreasing p62, upon CCCP treatment. A , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). B , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗∗ p < 0.001). C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM CCCP for the indicated times. Cell lysates were immunoblotted with the specified antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). D , SH-SY5Y cells were treated with vehicle or 10 μM CCCP for 24 h and then treated with vehicle or 50 μM CQ for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗∗∗ p < 0.001). E , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 10 μM CCCP alone or in combination with 50 μM CQ for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The relative p62 levels compared to β-actin and the relative LC3-Ⅱ levels compared to LC3-Ⅰ were quantified. The presented data represent the mean ± SEM of three independent experiments (∗∗ p < 0.01; ∗∗∗ p < 0.001). FBW7, F-box and WD repeat domain–containing 7; PINK1, PTEN-induced kinase 1; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CQ, chloroquine.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Transfection, Control

FBW7β depletion promotes reduction in mitochondrial proteins upon CCCP treatment. A , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM CCCP for the indicated times. Cell lysates were immunoblotted with the specified antibodies. β-Actin was used as a control for equal protein loading. B , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). C , SH-SY5Y cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The cell lysates were immunoblotted with the specified antibodies. β-Actin was used as a control for equal protein loading. D , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). E , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates nonspecific bands. F , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). G , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 10 μM CCCP alone or in combination with 50 μM CQ for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates nonspecific bands. H , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CQ, chloroquine; FBW7, F-box and WD repeat domain–containing 7; MEF, mouse embryonic fibroblast; PINK1, PTEN-induced kinase 1.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β depletion promotes reduction in mitochondrial proteins upon CCCP treatment. A , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 10 μM CCCP for the indicated times. Cell lysates were immunoblotted with the specified antibodies. β-Actin was used as a control for equal protein loading. B , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). C , SH-SY5Y cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The cell lysates were immunoblotted with the specified antibodies. β-Actin was used as a control for equal protein loading. D , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). E , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates nonspecific bands. F , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). G , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 10 μM CCCP alone or in combination with 50 μM CQ for additional 24 h. The cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates nonspecific bands. H , relative levels of five mitochondrial proteins ( i.e., VDAC1, HSP60, COX4, Mfn2, and Tom20) compared with β-actin were quantified, and the data are presented as the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CQ, chloroquine; FBW7, F-box and WD repeat domain–containing 7; MEF, mouse embryonic fibroblast; PINK1, PTEN-induced kinase 1.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Control, Transfection

FBW7β depletion promotes CCCP-induced mitophagy via PINK1–Parkin pathway. A , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 20 μM CCCP for 24 h. The MMP in each cell was detected using JC-1 staining. Representative confocal images of JC-1 aggregates ( red ) and JC-1 monomers ( green ) are shown. The scale bars represent 5 μm. The MMP ( red / green fluorescence ratio) were quantified using Image J software and depicted as a scatter plot. Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). B , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The MMP of each sample was detected using JC-1 staining. Representative confocal images of JC-1 aggregates ( red ) and monomers ( green ) are shown. The scale bars represent 5 μm. The MMP values were quantified using Image J software and depicted as a scatter plot. Data are presented as the mean ± SEM of three independent experiments (n = 24; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 20 μM CCCP for 24 h. The mitophagy ( red ) and lysosome dyes ( green ) were then used to stain each sample using the mitophagy detection kit (Dojindo). Representative confocal images showing the colocalization of mitophagy ( red ) and lysosome ( green ) are presented. The scale bars represent 10 μm. The extent of mitophagic flux (fluorescence intensity of red ) were quantified using Image J software and depicted as a scatter plot . Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). D , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The mitophagy ( red ) and lysosome dyes ( green ) were then used to stain each sample. Representative confocal images showing the colocalization of mitophagy ( red ) and lysosome ( green ) are presented. The scale bars represent 10 μm. The extent of mitophagic flux (fluorescence intensity of red ) were quantified using Image J software and depicted as a scatter plot . Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗∗ p < 0.001; n.s, not significant). E , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a control for equal protein loading. F , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a control for equal protein loading. G , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 20 μM MG132 for 30 min prior to addition of CCCP. Cells were then treated with vehicle or 20 μM CCCP for additional 3 h. Cell lysates were immunoprecipitated with anti-Mfn2 antibody, followed by immunoblotting with the indicated antibodies. The asterisk indicates IgG heavy chains. β-Actin served as a loading control. CCCP, carbonyl cyanide 3-chlorophenylhydrazone; FBW7, F-box and WD repeat domain–containing 7; MEF, mouse embryonic fibroblast; PINK1, PTEN-induced kinase 1.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β depletion promotes CCCP-induced mitophagy via PINK1–Parkin pathway. A , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 20 μM CCCP for 24 h. The MMP in each cell was detected using JC-1 staining. Representative confocal images of JC-1 aggregates ( red ) and JC-1 monomers ( green ) are shown. The scale bars represent 5 μm. The MMP ( red / green fluorescence ratio) were quantified using Image J software and depicted as a scatter plot. Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). B , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The MMP of each sample was detected using JC-1 staining. Representative confocal images of JC-1 aggregates ( red ) and monomers ( green ) are shown. The scale bars represent 5 μm. The MMP values were quantified using Image J software and depicted as a scatter plot. Data are presented as the mean ± SEM of three independent experiments (n = 24; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). C , FBW7 +/+ and FBW7 −/− HCT116 cells were treated with vehicle or 20 μM CCCP for 24 h. The mitophagy ( red ) and lysosome dyes ( green ) were then used to stain each sample using the mitophagy detection kit (Dojindo). Representative confocal images showing the colocalization of mitophagy ( red ) and lysosome ( green ) are presented. The scale bars represent 10 μm. The extent of mitophagic flux (fluorescence intensity of red ) were quantified using Image J software and depicted as a scatter plot . Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s, not significant). D , PINK1 +/+ and PINK1 −/− MEF cells were transfected for 48 h with scrambled control siRNA or FBW7β -siRNA and treated with vehicle or 10 μM CCCP for additional 24 h. The mitophagy ( red ) and lysosome dyes ( green ) were then used to stain each sample. Representative confocal images showing the colocalization of mitophagy ( red ) and lysosome ( green ) are presented. The scale bars represent 10 μm. The extent of mitophagic flux (fluorescence intensity of red ) were quantified using Image J software and depicted as a scatter plot . Data are presented as the mean ± SEM of three independent experiments (n = 45; ∗∗∗ p < 0.001; n.s, not significant). E , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a control for equal protein loading. F , SH-SY5Y cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with vehicle or 10 μM CCCP for additional 24 h. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a control for equal protein loading. G , HEK293 cells were transfected with scrambled control siRNA or FBW7β -siRNA for 48 h and treated with 20 μM MG132 for 30 min prior to addition of CCCP. Cells were then treated with vehicle or 20 μM CCCP for additional 3 h. Cell lysates were immunoprecipitated with anti-Mfn2 antibody, followed by immunoblotting with the indicated antibodies. The asterisk indicates IgG heavy chains. β-Actin served as a loading control. CCCP, carbonyl cyanide 3-chlorophenylhydrazone; FBW7, F-box and WD repeat domain–containing 7; MEF, mouse embryonic fibroblast; PINK1, PTEN-induced kinase 1.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Staining, Fluorescence, Software, Transfection, Control, Immunoprecipitation, Western Blot

FBW7β promotes staurosporine-induced cell death via PINK1 proteolysis. A , HEK293 cells were transfected with plasmid encoding PINK1-Myc for 24 h and treated with the indicated concentrations of STS for an additional 6 h. Cell lysates were immunoblotted using an anti-Myc antibody. Tubulin served as a loading control. B , HEK293 cells were sequentially transfected for 24 h with nonspecific control siRNA or FBW7β -siRNA and with PINK1-Myc for additional 24 h. Cells were then treated with either vehicle or 1 μM STS for 6 h. Cell lysates were immunoblotted with anti-Myc antibody. β-Actin served as a loading control. The relative PINK1 levels compared to β-actin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). C , where indicated, HEK293 cells were mock-transfected or transfected with plasmid encoding PINK1-V5-His for 24 h and treated sequentially with vehicle or 1 μM STS for 8 h and with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. β-Actin served as a loading control. D , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-V5-His, FLAG-FBW7β-WT, or FLAG-FBW7β-ΔF alone or in combination. Cells were then treated with vehicle or 1 μM STS for 8 h and treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. β-Actin served as a loading control. E , HEK293 cells were sequentially transfected for 24 h with nonspecific control siRNA or FBW7β -siRNA and with PINK1-V5-His for additional 24 h. Cells were then treated with vehicle or 1 μM STS for 8 h and with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. The asterisk indicates IgG heavy chains. β-Actin served as a loading control. F , SH-SY5Y cells were either mock-transfected or transfected with plasmids encoding Myc-PINK1 or/and FLAG-FBW7β for 24 h and treated with vehicle or 1 μM STS for an additional 24 h. Cell toxicity was measured using an LDH assay kit. The data are presented as the mean ± S.D. of four independent experiments (n = 4; ∗, p < 0.05; ∗∗∗, p < 0.001). G , SH-SY5Y cells were transfected with nonspecific control siRNA, FBW7β -siRNA, or PINK1 -siRNA alone or in combination for 48 h and treated with vehicle or 1 μM STS for an additional 24 h. Cell toxicity was measured using an LDH assay kit. The data are presented as the mean ± S.D. of four independent experiments (n = 4; ∗, p < 0.05; ∗∗∗, p < 0.001). FBW7, F-box and WD repeat domain–containing 7; LDH, lactate dehydrogenase; PINK1, PTEN-induced kinase 1; PLA, proximity ligation assay; STS, staurosporine.

Journal: The Journal of Biological Chemistry

Article Title: The SCF-FBW7β E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1

doi: 10.1016/j.jbc.2024.107198

Figure Lengend Snippet: FBW7β promotes staurosporine-induced cell death via PINK1 proteolysis. A , HEK293 cells were transfected with plasmid encoding PINK1-Myc for 24 h and treated with the indicated concentrations of STS for an additional 6 h. Cell lysates were immunoblotted using an anti-Myc antibody. Tubulin served as a loading control. B , HEK293 cells were sequentially transfected for 24 h with nonspecific control siRNA or FBW7β -siRNA and with PINK1-Myc for additional 24 h. Cells were then treated with either vehicle or 1 μM STS for 6 h. Cell lysates were immunoblotted with anti-Myc antibody. β-Actin served as a loading control. The relative PINK1 levels compared to β-actin were quantified. The presented data represent the mean ± SEM of three independent experiments (∗ p < 0.05; ∗∗ p < 0.01). C , where indicated, HEK293 cells were mock-transfected or transfected with plasmid encoding PINK1-V5-His for 24 h and treated sequentially with vehicle or 1 μM STS for 8 h and with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. β-Actin served as a loading control. D , HEK293 cells were transfected for 24 h with plasmids encoding PINK1-V5-His, FLAG-FBW7β-WT, or FLAG-FBW7β-ΔF alone or in combination. Cells were then treated with vehicle or 1 μM STS for 8 h and treated with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. β-Actin served as a loading control. E , HEK293 cells were sequentially transfected for 24 h with nonspecific control siRNA or FBW7β -siRNA and with PINK1-V5-His for additional 24 h. Cells were then treated with vehicle or 1 μM STS for 8 h and with 20 μM MG132 for additional 4 h. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with the specified antibodies. The asterisk indicates IgG heavy chains. β-Actin served as a loading control. F , SH-SY5Y cells were either mock-transfected or transfected with plasmids encoding Myc-PINK1 or/and FLAG-FBW7β for 24 h and treated with vehicle or 1 μM STS for an additional 24 h. Cell toxicity was measured using an LDH assay kit. The data are presented as the mean ± S.D. of four independent experiments (n = 4; ∗, p < 0.05; ∗∗∗, p < 0.001). G , SH-SY5Y cells were transfected with nonspecific control siRNA, FBW7β -siRNA, or PINK1 -siRNA alone or in combination for 48 h and treated with vehicle or 1 μM STS for an additional 24 h. Cell toxicity was measured using an LDH assay kit. The data are presented as the mean ± S.D. of four independent experiments (n = 4; ∗, p < 0.05; ∗∗∗, p < 0.001). FBW7, F-box and WD repeat domain–containing 7; LDH, lactate dehydrogenase; PINK1, PTEN-induced kinase 1; PLA, proximity ligation assay; STS, staurosporine.

Article Snippet: Polyclonal rabbit anti-PINK1 (BC100-494) antibody was obtained from Novus Biologicals.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Lactate Dehydrogenase Assay, Proximity Ligation Assay

Journal: Neural Regeneration Research

Article Title: Stress increases MHC-I expression in dopaminergic neurons and induces autoimmune activation in Parkinson's disease

doi: 10.4103/1673-5374.313057

Figure Lengend Snippet: The sequences of PINK1 gene

Article Snippet: The following primary antibodies were used for western blotting: rabbit polyclonal antibody against human PINK1 protein (BC100-494; Novus Biologicals, Centennial, CO, USA) and mouse monoclonal antibody against human glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab8245; Abcam).

Techniques: Negative Control

PINK1 is related to MHC-I expression in SH-SY5Y cells. (A) Flow cytometric analysis of MHC-I expression in neuron membranes. (B) Quantification of the flow cytometric analysis results. Data are expressed as the mean ± SEM ( n = 6; one-way analysis of variance followed by Bonferroni post hoc test). * P < 0.05. (C) Western blot assay of PINK1 expression in SH-SY5Y cells that had been transfected with control or PINK1 siRNA. (D) Fluorescence images showing the expression of MHC-1 (green) in SH-SY5Y cells. Scale bar: 5 μm. The arrow indicates MHC-I protein presenting on the cell surface. MHC-I: Major histocompatibility complex class I; MPP + : 1-methyl-4-phenylpyridinium; PINK1: PTEN-induced kinase 1.

Journal: Neural Regeneration Research

Article Title: Stress increases MHC-I expression in dopaminergic neurons and induces autoimmune activation in Parkinson's disease

doi: 10.4103/1673-5374.313057

Figure Lengend Snippet: PINK1 is related to MHC-I expression in SH-SY5Y cells. (A) Flow cytometric analysis of MHC-I expression in neuron membranes. (B) Quantification of the flow cytometric analysis results. Data are expressed as the mean ± SEM ( n = 6; one-way analysis of variance followed by Bonferroni post hoc test). * P < 0.05. (C) Western blot assay of PINK1 expression in SH-SY5Y cells that had been transfected with control or PINK1 siRNA. (D) Fluorescence images showing the expression of MHC-1 (green) in SH-SY5Y cells. Scale bar: 5 μm. The arrow indicates MHC-I protein presenting on the cell surface. MHC-I: Major histocompatibility complex class I; MPP + : 1-methyl-4-phenylpyridinium; PINK1: PTEN-induced kinase 1.

Techniques: Expressing, Western Blot, Transfection, Control, Fluorescence, Immunopeptidomics

(A) Western blots of PINK1, Parkin, and MFN2 immunoreactivities. (B) Total membrane protein detected by SYPRO Ruby staining. (C-F) Comparisons of the immunoreactivities of PINK1, 50 kDa Parkin, approximately 40 kDa Parkin, and MFN2 expression normalized to total protein between groups are indicated in C, D, E, and F, respectively. (G) Comparison of relative MFN2 level calculated from each MFN2 band intensity normalized to total MFN2 is expressed in G as a percentage. CON, control. HU, hindlimb unloading. Data are expressed as means ± SE. **significantly different from CON (p < 0.01).

Journal: PLoS ONE

Article Title: Potential roles of neuronal nitric oxide synthase and the PTEN-induced kinase 1 (PINK1)/Parkin pathway for mitochondrial protein degradation in disuse-induced soleus muscle atrophy in adult rats

doi: 10.1371/journal.pone.0243660

Figure Lengend Snippet: (A) Western blots of PINK1, Parkin, and MFN2 immunoreactivities. (B) Total membrane protein detected by SYPRO Ruby staining. (C-F) Comparisons of the immunoreactivities of PINK1, 50 kDa Parkin, approximately 40 kDa Parkin, and MFN2 expression normalized to total protein between groups are indicated in C, D, E, and F, respectively. (G) Comparison of relative MFN2 level calculated from each MFN2 band intensity normalized to total MFN2 is expressed in G as a percentage. CON, control. HU, hindlimb unloading. Data are expressed as means ± SE. **significantly different from CON (p < 0.01).

Article Snippet: The primary antibodies used in the present study were as follows: rabbit anti-nNOS monoclonal antibody (C7D7, #4231, Cell Signaling Technology, diluted 1:1000), rabbit anti-nNOS (phospho S1417) polyclonal antibody (ab5583, Abcam, diluted 1:2000), rabbit anti-nNOS (phospho S847) polyclonal antibody (ab16650, Abcam, diluted 1:4000), rabbit anti-calmodulin polyclonal antibody (#4830, Cell Signaling Technology, diluted 1:1000), rabbit anti-HSP90 polyclonal antibody (ab13495, Abcam, diluted 1:1000), mouse anti-HSP70 monoclonal antibody (C92F3A-5, ADI-SPA-810, Enzo Life Sciences, diluted 1:1000), mouse anti-malondialdehyde (MDA) monoclonal antibody (MMD-030n, Japan Institute for the Control of Aging, NIKKEN SEIL CO., Ltd, diluted 1:200), mouse anti-6-nitrotryptophan (6-NO 2 Trp) monoclonal antibody (Japan Institute for the Control of Aging, NIKKEN SEIL CO., Ltd, 1μg/ml), mouse anti-nitrotyrosine (3-NT) monoclonal antibody (39B6, SC-32757, Santa Cruz Biotechnology, Inc. diluted 1:1000), mouse anti-nitrosocystein monoclonal antibody (HY8E12, ab94930, abcam, diluted 1:1000), rabbit anti-PINK1 polyclonal antibody (BC100-494, Novus Biologicals, diluted 1:2000), mouse anti-Parkin monoclonal antibody (PRK8, ab77924, Abcam, diluted 1:1000), mouse anti-MFN2 monoclonal antibody (6A8, ab56889, Abcam, diluted 1:1000), and mouse anti-mono- and polyubiquitinylated conjugates monoclonal antibody (FK2, BML-PW8810-0100, Enzo, diluted 1:1000).

Techniques: Western Blot, Membrane, Staining, Expressing, Comparison, Control

Confocal microscopy analysis of the mitophagy initiation in the RPE cells by staining PINK1 and PARKIN. One-year-old WT and dKO mice focusing on the RPE cells in the vicinity of the optic nerve ( a , e ). PINK1 ( b , red) and PARKIN ( c , green) were double-stained and the merged image ( d ) was used to count the colocalized puncta from WT. Similarly, in dKO PINK1 ( f , red) and PARKIN ( g , green) were double-stained, and the merged image ( h ) was used to count the colocalized puncta. In dKO, we observed a ~7% decrease in the total number of puncta; however, the number of colocalizations was increased by ~118% ( i ). Scale = 5 µm. *p = 0.01.

Journal: International Journal of Molecular Sciences

Article Title: Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2 / PGC-1α -/- Mouse Model

doi: 10.3390/ijms21061976

Figure Lengend Snippet: Confocal microscopy analysis of the mitophagy initiation in the RPE cells by staining PINK1 and PARKIN. One-year-old WT and dKO mice focusing on the RPE cells in the vicinity of the optic nerve ( a , e ). PINK1 ( b , red) and PARKIN ( c , green) were double-stained and the merged image ( d ) was used to count the colocalized puncta from WT. Similarly, in dKO PINK1 ( f , red) and PARKIN ( g , green) were double-stained, and the merged image ( h ) was used to count the colocalized puncta. In dKO, we observed a ~7% decrease in the total number of puncta; however, the number of colocalizations was increased by ~118% ( i ). Scale = 5 µm. *p = 0.01.

Article Snippet: The mouse monoclonal antibody PARKIN (sc-32282, Santa Cruz Biologicals, Dallas, TX, USA) and rabbit polyclonal PINK1 (BC100-494, Novus Biologicals, Briarwood Avenue, Building IV Centennial, CO, USA) antibodies were applied to detect respective epitopes.

Techniques: Confocal Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2 / PGC-1α -/- Mouse Model

doi: 10.3390/ijms21061976

Figure Lengend Snippet: List of primary antibodies.

Techniques:

Review

Structured Review

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