phospho atg13 s355 d6j1w  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc phospho atg13 s355 d6j1w
    Phospho Atg13 S355 D6j1w, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho atg13 s355 d6j1w/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho atg13 s355 d6j1w - by Bioz Stars, 2023-02
    94/100 stars

    Images

    phospho atg13 s355 d6j1w  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc phospho atg13 s355 d6j1w
    Phospho Atg13 S355 D6j1w, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho atg13 s355 d6j1w/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho atg13 s355 d6j1w - by Bioz Stars, 2023-02
    94/100 stars

    Images

    p atg13  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc p atg13
    Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), <t>p-Atg13</t> (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    P Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p atg13 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues"

    Article Title: Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

    Journal: Journal of Virology

    doi: 10.1128/JVI.01537-21

    Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Infection, Western Blot, Quantitative RT-PCR, Transfection

    phosphor atg13 ser355  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc phosphor atg13 ser355
    Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, <t>ATG13,</t> ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).
    Phosphor Atg13 Ser355, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphor atg13 ser355/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphor atg13 ser355 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "ω-6 Polyunsaturated fatty acids (linoleic acid) activate both autophagy and antioxidation in a synergistic feedback loop via TOR-dependent and TOR-independent signaling pathways"

    Article Title: ω-6 Polyunsaturated fatty acids (linoleic acid) activate both autophagy and antioxidation in a synergistic feedback loop via TOR-dependent and TOR-independent signaling pathways

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-02750-0

    Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, ATG13, ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).
    Figure Legend Snippet: Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, ATG13, ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Techniques Used: CCK-8 Assay, Expressing, Western Blot, Staining, Flow Cytometry, Fluorescence

    The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 μM MHY1485) ( n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptor Ser792 , T-TSC2, P-TSC2 Ser1387 , T-ULK1, P-ULK1 Ser467 , P-ULK1 Ser555 , P-ULK1 Ser637 , P-ULK1 Ser757 , T-ATG13, P-ATG13 Ser355 , T-TFEB, P-TFEB Ser211 , and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls ( a , b ). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 μm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group ( c , e ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator ( d , f ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator ( n = 6) ( g , h ). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).
    Figure Legend Snippet: The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 μM MHY1485) ( n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptor Ser792 , T-TSC2, P-TSC2 Ser1387 , T-ULK1, P-ULK1 Ser467 , P-ULK1 Ser555 , P-ULK1 Ser637 , P-ULK1 Ser757 , T-ATG13, P-ATG13 Ser355 , T-TFEB, P-TFEB Ser211 , and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls ( a , b ). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 μm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group ( c , e ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator ( d , f ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator ( n = 6) ( g , h ). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Techniques Used: Western Blot, Staining, Flow Cytometry, Fluorescence, Expressing

    anti atg13 s355  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc anti atg13 s355
    (A) <t>ULK1-ATG13</t> shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.
    Anti Atg13 S355, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti atg13 s355/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti atg13 s355 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle"

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000288

    (A) ULK1-ATG13 shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.
    Figure Legend Snippet: (A) ULK1-ATG13 shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.

    Techniques Used: Mobility Shift, SDS Page, Western Blot, Electrophoretic Mobility Shift Assay, Staining, Blocking Assay, Immunoprecipitation

    (A-B) ULK1-ATG13 upshifted band in mitosis could be recognized by CDK substrate-specific antibodies. The 293T cells stably expressing FLAG-tagged mULK1 or ATG13 were synchronized by single-thymidine and released in the presence or absence of nocodazole. The coimmunoprecipitates and input were immunoblotted with specific antibodies. The phospho-signal was quantified relative to the FLAG antibody–detected bands (mULK1/ATG13-3FLAG) in IP; the IP signal of mULK1/ATG13-3FLAG was quantified relative to its input. Various cell cycle markers were detected to show the respective phases of cell cycle. ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200 kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; p-MAPK, phospho mitogen-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1.
    Figure Legend Snippet: (A-B) ULK1-ATG13 upshifted band in mitosis could be recognized by CDK substrate-specific antibodies. The 293T cells stably expressing FLAG-tagged mULK1 or ATG13 were synchronized by single-thymidine and released in the presence or absence of nocodazole. The coimmunoprecipitates and input were immunoblotted with specific antibodies. The phospho-signal was quantified relative to the FLAG antibody–detected bands (mULK1/ATG13-3FLAG) in IP; the IP signal of mULK1/ATG13-3FLAG was quantified relative to its input. Various cell cycle markers were detected to show the respective phases of cell cycle. ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200 kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; p-MAPK, phospho mitogen-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation

    (A) Mitotic ATG13 undergoes mobility upshift in mitosis in electrophoresis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine in the presence or absence of nocodazole and coimmunoprecipitated by the FLAG antibody followed by western blot analysis. Size of endogenous ATG and expressed FLAG-tagged ATG13 were not distinguishable in electrophoresis here. (B) The effect of AMPK inhibition on ULK1 and ATG13 mobility shift in mitosis. The AMPK inhibitor Compound C was used to evaluate the role of AMPK on ULK1 or ATG13 mobility shift as . Phospho-AMPKα-T172 and phospho-Acetyl-CoA Carboxylase (Ser79) were used to indicate AMPK inhibition. (C) CDK1 inhibitor RO-3306 decreases the ATG13 band shift in mitosis. HeLa cells synchronized and treated as were subjected to western blot analysis. (D) ATG13 is phosphorylated and interacts with CDK1/cyclin B1 in mitosis. The 293T cells overexpressing FLAG-tagged ATG13 were synchronized by single-thymidine in the presence or absence of nocodazole. The coimmunoprecipitates by FLAG antibody were subjected to western blot analysis. (E-F) ATG13 is directly phosphorylated by purified CDK1/cyclin B complex in vitro. The ATG13 immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged ATG13 or ULK1 and purified CDK1/cyclin B complex were subjected to in vitro kinase assay with or without RO-3306 followed by western blot analysis. (E) shows representative western blot analysis of the ATG13 immunoprecipitates as substrate, and (F) shows the representative western blot analysis of the ULK1-WT coimmunoprecipitates as substrate. AMPK, AMP-activated protein kinase; ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.
    Figure Legend Snippet: (A) Mitotic ATG13 undergoes mobility upshift in mitosis in electrophoresis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine in the presence or absence of nocodazole and coimmunoprecipitated by the FLAG antibody followed by western blot analysis. Size of endogenous ATG and expressed FLAG-tagged ATG13 were not distinguishable in electrophoresis here. (B) The effect of AMPK inhibition on ULK1 and ATG13 mobility shift in mitosis. The AMPK inhibitor Compound C was used to evaluate the role of AMPK on ULK1 or ATG13 mobility shift as . Phospho-AMPKα-T172 and phospho-Acetyl-CoA Carboxylase (Ser79) were used to indicate AMPK inhibition. (C) CDK1 inhibitor RO-3306 decreases the ATG13 band shift in mitosis. HeLa cells synchronized and treated as were subjected to western blot analysis. (D) ATG13 is phosphorylated and interacts with CDK1/cyclin B1 in mitosis. The 293T cells overexpressing FLAG-tagged ATG13 were synchronized by single-thymidine in the presence or absence of nocodazole. The coimmunoprecipitates by FLAG antibody were subjected to western blot analysis. (E-F) ATG13 is directly phosphorylated by purified CDK1/cyclin B complex in vitro. The ATG13 immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged ATG13 or ULK1 and purified CDK1/cyclin B complex were subjected to in vitro kinase assay with or without RO-3306 followed by western blot analysis. (E) shows representative western blot analysis of the ATG13 immunoprecipitates as substrate, and (F) shows the representative western blot analysis of the ULK1-WT coimmunoprecipitates as substrate. AMPK, AMP-activated protein kinase; ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Techniques Used: Electrophoresis, Western Blot, Inhibition, Mobility Shift, Electrophoretic Mobility Shift Assay, Purification, In Vitro, Kinase Assay, Immunoprecipitation

    (A) The mitotic-specific phosphorylated sites identified by MS in mitotic mULK1 compared with asynchronous mULK1. (B-C) S622/T635/T653 phosphorylation contributes to ULK1 mobility shift in mitosis. The 293T cells overexpressing FLAG-tagged mULK1-S622/T635/T653 mutants were synchronized with single-thymidine and nocodazole. Then immunoprecipitation for single mutant with FLAG antibody (B) or western blot analysis for double and triple mutant (C) was performed. (D-E) More sites contribute to ULK1 band shift. Based on triple 3A mutant, the other 8 Ser/Thr sites were mutated into Ala. The 293T cells expressing various mutants were synchronized into mitosis with thymidine and nocodazole for immunoprecipitation by FLAG antibody and western blot analysis. (F) ULK1-11A mutant was not upshifted or phosphorylated by CDK1/cyclin B kinase complex in vitro. The ULK1-11A mutant immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged mULK1 and purified CDK1/cyclin B complex were tested in an in vitro kinase assay and western blot analysis. (G) The phosphorylated sites identified by MS in mitotic ATG13 compared with asynchronous ATG13. (H-I) ATG13-T342/T332/S44/S224 phosphorylation contributes to ATG13 mobility shift in mitosis. HeLa-ATG13 KO cells (H) or 293T cells (I) overexpressing FLAG-tagged ATG13-T342/T332/S44/S224 mutants were synchronized with single-thymidine and nocodazole. Then western blot analysis for 4-site mutant (H) or immunoprecipitation and input for mutants with FLAG antibody (I) was performed. 3A, S622/T635/T653A; 5A, 3A-S479&S543A; 7A, 5A-S411&S413A; 9A, 5A-S413&T401&S403&S405A; 10A, 9A-T282A; 11A, 10A-T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; GFP, green fluorescent protein; IP, immunoprecipitation; MS, mass spectrometry; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.
    Figure Legend Snippet: (A) The mitotic-specific phosphorylated sites identified by MS in mitotic mULK1 compared with asynchronous mULK1. (B-C) S622/T635/T653 phosphorylation contributes to ULK1 mobility shift in mitosis. The 293T cells overexpressing FLAG-tagged mULK1-S622/T635/T653 mutants were synchronized with single-thymidine and nocodazole. Then immunoprecipitation for single mutant with FLAG antibody (B) or western blot analysis for double and triple mutant (C) was performed. (D-E) More sites contribute to ULK1 band shift. Based on triple 3A mutant, the other 8 Ser/Thr sites were mutated into Ala. The 293T cells expressing various mutants were synchronized into mitosis with thymidine and nocodazole for immunoprecipitation by FLAG antibody and western blot analysis. (F) ULK1-11A mutant was not upshifted or phosphorylated by CDK1/cyclin B kinase complex in vitro. The ULK1-11A mutant immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged mULK1 and purified CDK1/cyclin B complex were tested in an in vitro kinase assay and western blot analysis. (G) The phosphorylated sites identified by MS in mitotic ATG13 compared with asynchronous ATG13. (H-I) ATG13-T342/T332/S44/S224 phosphorylation contributes to ATG13 mobility shift in mitosis. HeLa-ATG13 KO cells (H) or 293T cells (I) overexpressing FLAG-tagged ATG13-T342/T332/S44/S224 mutants were synchronized with single-thymidine and nocodazole. Then western blot analysis for 4-site mutant (H) or immunoprecipitation and input for mutants with FLAG antibody (I) was performed. 3A, S622/T635/T653A; 5A, 3A-S479&S543A; 7A, 5A-S411&S413A; 9A, 5A-S413&T401&S403&S405A; 10A, 9A-T282A; 11A, 10A-T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; GFP, green fluorescent protein; IP, immunoprecipitation; MS, mass spectrometry; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Techniques Used: Mobility Shift, Immunoprecipitation, Mutagenesis, Western Blot, Electrophoretic Mobility Shift Assay, Expressing, In Vitro, Purification, Kinase Assay, Mass Spectrometry

    (A) The localization of mutant ULK1-11A and ATG13-4A in mitosis. Cells released from thymidine block for 10 hours were fixed with 3.7% formaldehyde for 20 minutes at room temperature and then for blocking and FLAG antibody, Alexa-488 conjugated secondary antibody, DAPI staining. Scale bar, 10 μm. (B) The ULK1 kinase activity is not affected by the 11A mutation. ATG13-Ser355 is the substrate of ULK1, which corresponds to Ser318 of ATG13 isoform 2. ULK1-KO 293T cells reconstituted with ULK1-WT, ULK1-11A, or ULK1-K46I were plated and lysed with M-PER. The western blot for ATG13-Ser355 indicated that ULK1 activity was not affected by 11A mutation. n = 3. (C) Autophagy activity was examined in ULK1&ATG13 double WT or mutant (“Mut”) cells expressing GFP-LC3-RFP by flow cytometry. ULK1&ATG13 double WT or mutant cells, HeLa, or HeLa- ULK1&ATG13 DKO cells stably expressing GFP-LC3-RFP were established by infection of retrovirus packaged by pMRX-IP-GFP-LC3-RFP and helper plasmids Vsvg and pMLV. Mitotic cells were collected by shake-off from thymidine and nocodazole synchronized cells for flow cytometry detection. The GFP/RFP ratio inversely correlated with autophagy activity. n = 4, *** p < 0.001, **** p < 0.0001. (D) The autophagic flux of HeLa-DKO cells stably overexpressing double WT or mutant FLAG-tagged mULK1 and ATG13. Cells synchronized to mitosis with thymidine and nocodazole were shaken off and treated with or without autophagy inhibitor 25 μM CQ for 1 hour. The left panel is a representative western blot, and the right panel is the statistical result for LC3B-II and p62. n = 3, * p < 0.05, ** p < 0.01. (E) LC3B puncta number was counted, and the micrographs were captured by Zeiss LSM710 confocal microscope. Scale bar, 10 μm. n = 43, ** p < 0.01. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CQ, chloroquine; Ctrl, control; DAPI, 4 DAPdiamidino-2-phenylindole; DKO, double knockout; GFP, green fluorescent protein; KO, knockout; M-PER, Mammalian Protein Extraction Reagent; mULK1, mouse ULK1; n.s., not significant; RFP, red fluorescent protein; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.
    Figure Legend Snippet: (A) The localization of mutant ULK1-11A and ATG13-4A in mitosis. Cells released from thymidine block for 10 hours were fixed with 3.7% formaldehyde for 20 minutes at room temperature and then for blocking and FLAG antibody, Alexa-488 conjugated secondary antibody, DAPI staining. Scale bar, 10 μm. (B) The ULK1 kinase activity is not affected by the 11A mutation. ATG13-Ser355 is the substrate of ULK1, which corresponds to Ser318 of ATG13 isoform 2. ULK1-KO 293T cells reconstituted with ULK1-WT, ULK1-11A, or ULK1-K46I were plated and lysed with M-PER. The western blot for ATG13-Ser355 indicated that ULK1 activity was not affected by 11A mutation. n = 3. (C) Autophagy activity was examined in ULK1&ATG13 double WT or mutant (“Mut”) cells expressing GFP-LC3-RFP by flow cytometry. ULK1&ATG13 double WT or mutant cells, HeLa, or HeLa- ULK1&ATG13 DKO cells stably expressing GFP-LC3-RFP were established by infection of retrovirus packaged by pMRX-IP-GFP-LC3-RFP and helper plasmids Vsvg and pMLV. Mitotic cells were collected by shake-off from thymidine and nocodazole synchronized cells for flow cytometry detection. The GFP/RFP ratio inversely correlated with autophagy activity. n = 4, *** p < 0.001, **** p < 0.0001. (D) The autophagic flux of HeLa-DKO cells stably overexpressing double WT or mutant FLAG-tagged mULK1 and ATG13. Cells synchronized to mitosis with thymidine and nocodazole were shaken off and treated with or without autophagy inhibitor 25 μM CQ for 1 hour. The left panel is a representative western blot, and the right panel is the statistical result for LC3B-II and p62. n = 3, * p < 0.05, ** p < 0.01. (E) LC3B puncta number was counted, and the micrographs were captured by Zeiss LSM710 confocal microscope. Scale bar, 10 μm. n = 43, ** p < 0.01. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CQ, chloroquine; Ctrl, control; DAPI, 4 DAPdiamidino-2-phenylindole; DKO, double knockout; GFP, green fluorescent protein; KO, knockout; M-PER, Mammalian Protein Extraction Reagent; mULK1, mouse ULK1; n.s., not significant; RFP, red fluorescent protein; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Techniques Used: Mutagenesis, Blocking Assay, Staining, Activity Assay, Western Blot, Expressing, Flow Cytometry, Stable Transfection, Infection, Microscopy, Double Knockout, Knock-Out, Protein Extraction

    (A) ULK1-ATG13 DKO inhibits S/G2 and G2/M transitions. HeLa WT or ULK1&ATG13-DKO cells synchronized into mitosis were subjected to PI and pH3(S10) co-staining for cell cycle and mitotic index analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (B) Representative western blot analysis suggests that ULK1&ATG13 DKO inhibits mitotic entry, which is shown by mitotic markers and CDK1 substrate phosphorylation. (C) Mitotic exit of ULK1, ATG13, or ULK1&ATG13 KO cells. Cells were synchronized into mitosis with thymidine and NOC and released into NOC-free complete DMEM medium for different timepoints and then subjected to either PI and pH3(S10) co-staining or cyclin B1 staining for cell cycle, mitotic index, and cyclin B1 level analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (D) ULK1&ATG13 DKO inhibits cell proliferation. HeLa WT or ULK1&ATG13 DKO cells were plated at 1 × 10 5 cells/mL and cultured for 1, 2, or 3 days. The cell number was counted by flow cytometry and the doubling time was calculated. T D indicates the average cell doubling time and is calculated as: T D = t * [lg2/(lgNt − lgN0)], where t is the culture time, Nt is the cell number after culturing, and N0 is the original cell number plated. n = 3, * p < 0.05, *** p < 0.001. (E-G) The role of ULK1-ATG13 phosphorylation in cell cycle progression and cell proliferation. ULK1&ATG13-DKO cells reconstituted with WT mULK1-3FLAG and ATG13-3FLAG or mutant (“Mut”) mULK1-11A and ATG13-4A, HeLa, or ULK1&ATG13-DKO cells were treated as (A)/(B) and (D). The mitotic exit (G1%) is the percentage of cells in G1 phase when released for indicated time. n = 3, ** p < 0.01, *** p < 0.001. (H) The relative tumor volume growth curve of nude mice bearing different tumors with or without SBI-0206965. The nude mice were injected with cells (1 × 10 7 ) in 100 μL PBS/Matrigel Matrix (1:1). Seven days postimplantation, 5 mice in each group were injected with 0.5% (M/V) methyl cellulose or SBI-0206965 in 0.5% methyl cellulose (20 mg/kg/d) every day for 33 days. Tumor growth was evaluated every day and tumor volume was calculated as: volume = 1/2 (length × width 2 ). (I) Tumor growth in nude mice bearing WT or KO cells with or without ULK1 kinase inhibitor SBI-0206965. The protocols were indicated as and the mice were sacrificed, and tumors were harvested and weighed up at the end of the experiment. n = 5, * p < 0.05, *** p < 0.001. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; DKO, double knockout; DMEM, Dulbecco’s Modified Eagle Medium; KO, knockout; mULK1, mouse ULK1; Myt1, myelin transcription factor 1; NOC, nocodazole; n.s., not significant; pH3(S10), phospho histone H3 serine 10; PI, propidium iodide; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.
    Figure Legend Snippet: (A) ULK1-ATG13 DKO inhibits S/G2 and G2/M transitions. HeLa WT or ULK1&ATG13-DKO cells synchronized into mitosis were subjected to PI and pH3(S10) co-staining for cell cycle and mitotic index analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (B) Representative western blot analysis suggests that ULK1&ATG13 DKO inhibits mitotic entry, which is shown by mitotic markers and CDK1 substrate phosphorylation. (C) Mitotic exit of ULK1, ATG13, or ULK1&ATG13 KO cells. Cells were synchronized into mitosis with thymidine and NOC and released into NOC-free complete DMEM medium for different timepoints and then subjected to either PI and pH3(S10) co-staining or cyclin B1 staining for cell cycle, mitotic index, and cyclin B1 level analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (D) ULK1&ATG13 DKO inhibits cell proliferation. HeLa WT or ULK1&ATG13 DKO cells were plated at 1 × 10 5 cells/mL and cultured for 1, 2, or 3 days. The cell number was counted by flow cytometry and the doubling time was calculated. T D indicates the average cell doubling time and is calculated as: T D = t * [lg2/(lgNt − lgN0)], where t is the culture time, Nt is the cell number after culturing, and N0 is the original cell number plated. n = 3, * p < 0.05, *** p < 0.001. (E-G) The role of ULK1-ATG13 phosphorylation in cell cycle progression and cell proliferation. ULK1&ATG13-DKO cells reconstituted with WT mULK1-3FLAG and ATG13-3FLAG or mutant (“Mut”) mULK1-11A and ATG13-4A, HeLa, or ULK1&ATG13-DKO cells were treated as (A)/(B) and (D). The mitotic exit (G1%) is the percentage of cells in G1 phase when released for indicated time. n = 3, ** p < 0.01, *** p < 0.001. (H) The relative tumor volume growth curve of nude mice bearing different tumors with or without SBI-0206965. The nude mice were injected with cells (1 × 10 7 ) in 100 μL PBS/Matrigel Matrix (1:1). Seven days postimplantation, 5 mice in each group were injected with 0.5% (M/V) methyl cellulose or SBI-0206965 in 0.5% methyl cellulose (20 mg/kg/d) every day for 33 days. Tumor growth was evaluated every day and tumor volume was calculated as: volume = 1/2 (length × width 2 ). (I) Tumor growth in nude mice bearing WT or KO cells with or without ULK1 kinase inhibitor SBI-0206965. The protocols were indicated as and the mice were sacrificed, and tumors were harvested and weighed up at the end of the experiment. n = 5, * p < 0.05, *** p < 0.001. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; DKO, double knockout; DMEM, Dulbecco’s Modified Eagle Medium; KO, knockout; mULK1, mouse ULK1; Myt1, myelin transcription factor 1; NOC, nocodazole; n.s., not significant; pH3(S10), phospho histone H3 serine 10; PI, propidium iodide; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Techniques Used: Staining, Flow Cytometry, Western Blot, Cell Culture, Mutagenesis, Injection, Double Knockout, Modification, Knock-Out

    rabbit anti phospho atg13  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc rabbit anti phospho atg13
    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, <t>ATG13,</t> p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments
    Rabbit Anti Phospho Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho atg13 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "TOPK inhibits autophagy by phosphorylating ULK1 and promotes glioma resistance to TMZ"

    Article Title: TOPK inhibits autophagy by phosphorylating ULK1 and promotes glioma resistance to TMZ

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1805-9

    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments
    Figure Legend Snippet: a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments

    Techniques Used: Over Expression, Western Blot, SDS Page

    a PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. c PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Then cells were treated with EGF (20 ng/ml) for 15 min, followed by the addition of CHX (100 μg/ml) for 6, 8, and 12 h before extraction. HA and Tubulin were analyzed using western blots (left). Data of c represent the results of triplicate experiments. The densities of HA/Tubulin (right) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. d HA-ULK1-WT or HA-ULK1-AAA, Flag-ubiquitin, and GAL4-TOPK were cotransfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-Flag, anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. e PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Cells were harvested 48 h later. LC3B, HA, and Tubulin were analyzed using western blots. f PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. 24 h later, cells were starved for 24 h, then rapamycin (50 nM) and CQ (50 μM) were added, respectively, for 15 min and 1 h. LC3B, HA, and Tubulin were analyzed using western blots. The densities of LC3-II/Tubulin (Fig. 6 f , bottom) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. Data represent the results of triplicate experiments
    Figure Legend Snippet: a PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. c PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Then cells were treated with EGF (20 ng/ml) for 15 min, followed by the addition of CHX (100 μg/ml) for 6, 8, and 12 h before extraction. HA and Tubulin were analyzed using western blots (left). Data of c represent the results of triplicate experiments. The densities of HA/Tubulin (right) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. d HA-ULK1-WT or HA-ULK1-AAA, Flag-ubiquitin, and GAL4-TOPK were cotransfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-Flag, anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. e PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Cells were harvested 48 h later. LC3B, HA, and Tubulin were analyzed using western blots. f PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. 24 h later, cells were starved for 24 h, then rapamycin (50 nM) and CQ (50 μM) were added, respectively, for 15 min and 1 h. LC3B, HA, and Tubulin were analyzed using western blots. The densities of LC3-II/Tubulin (Fig. 6 f , bottom) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. Data represent the results of triplicate experiments

    Techniques Used: Transfection, Western Blot, Immunoprecipitation

    rabbit anti atg13 polyclonal antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc rabbit anti atg13 polyclonal antibody
    Rabbit Anti Atg13 Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti atg13 polyclonal antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti atg13 polyclonal antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    rabbit anti phospho atg13 ser355 antibody  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc rabbit anti phospho atg13 ser355 antibody
    Rabbit Anti Phospho Atg13 Ser355 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho atg13 ser355 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho atg13 ser355 antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    rabbit anti atg13  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Cell Signaling Technology Inc rabbit anti atg13
    Identification of residues 582–585 LQFL in FIP200, required for its interaction with <t>Atg13</t> and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .
    Rabbit Anti Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti atg13 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model"

    Article Title: Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    Journal: Genes & Development

    doi: 10.1101/gad.276428.115

    Identification of residues 582–585 LQFL in FIP200, required for its interaction with Atg13 and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .
    Figure Legend Snippet: Identification of residues 582–585 LQFL in FIP200, required for its interaction with Atg13 and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .

    Techniques Used: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Molecular Weight

    The FIP200-4A mutant unable to bind ATG13 is defective in amino acid starvation-induced autophagy. ( A ) HEK293T cells were transfected with expression vectors encoding HA-tagged FIP200 and FIP200-4A mutant or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA followed by immunoblotting using anti-HA ( top ), anti-ULK1 ( middle ), or anti-Atg13 ( bottom ). ( B ) GFP-FIP200 ( left ) and GFP-FIP200-4A mutant puncta formation in starved HEK293T cells. ( C , D ) shRNA FIP200 knockdown HEK293T cells reconstituted with shRNA-resistant GFP-FIP200 or the GFP-FIP200-4A mutant were either left untreated or starved (Hank's balanced salt solution [HBSS]) for 2 h and then analyzed by immunoblotting with the indicated antibodies. ( C ) BafA1 (100 nM), an inhibitor of lysosome degradation, was included for 2 h in samples as indicated. ( D ) Quantification of LC3I-to-LC3II conversion in FIP200 knockdown HEK293T cells reconstituted with GFP-FIP200 and GFP-FIP200-4A upon starvation as shown in C . (**) P < 0.01.
    Figure Legend Snippet: The FIP200-4A mutant unable to bind ATG13 is defective in amino acid starvation-induced autophagy. ( A ) HEK293T cells were transfected with expression vectors encoding HA-tagged FIP200 and FIP200-4A mutant or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA followed by immunoblotting using anti-HA ( top ), anti-ULK1 ( middle ), or anti-Atg13 ( bottom ). ( B ) GFP-FIP200 ( left ) and GFP-FIP200-4A mutant puncta formation in starved HEK293T cells. ( C , D ) shRNA FIP200 knockdown HEK293T cells reconstituted with shRNA-resistant GFP-FIP200 or the GFP-FIP200-4A mutant were either left untreated or starved (Hank's balanced salt solution [HBSS]) for 2 h and then analyzed by immunoblotting with the indicated antibodies. ( C ) BafA1 (100 nM), an inhibitor of lysosome degradation, was included for 2 h in samples as indicated. ( D ) Quantification of LC3I-to-LC3II conversion in FIP200 knockdown HEK293T cells reconstituted with GFP-FIP200 and GFP-FIP200-4A upon starvation as shown in C . (**) P < 0.01.

    Techniques Used: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, shRNA

    Disruption of FIP200 interaction with Atg13 blocks autophagy in MEFs. ( A ) Schematic representation of the FIP200-4A mutant knock-in targeting vector and the targeted allele of the FIP200-4A mutant. Solid boxes represent regions of vector homology with the target locus; thick, numbered boxes represent the exons flanking homology regions. Site-directed mutagenesis was used to introduce the LQFL-to-AAAA mutation at the 582–585 amino acid residues. The asterisk denotes the mutated residues on exon 13. (EcoRV) EcoRV sites; (neo) neomycin-resistant gene cassette; (DTA) diphtheria toxin gene. P1 and P2 are a pair of primers for genotyping. P3 and P4 are a pair of primers for sequencing amplified genomic DNA with mutations. ( B ) Genomic DNA was extracted from mouse tails and analyzed by PCR using P1 and P2 to detect the FIP200 wild-type allele (178 base pairs [bp]) or knock-in allele (448 bp). Representative genotypes of a litter of mice from crosses of FIP200 +/ KI mice with wild-type B6 mice. ( C ) Representative genotypes of MEF cells prepared from embryonic day 13.5 (E13.5) embryos of intercrosses of FIP200 +/ KI mice. ( D ) Lysates from different MEFs were analyzed by immunoblotting using antibodies against various proteins as indicated. ( E ) MEFs were incubated in fresh growth medium (control) or HBSS (starvation) in the presence of 100 nM BafA1 for 2 h. Lysates were then prepared and analyzed by immunoblotting using antibodies against proteins as indicated. ( F ) FIP200 +/+ and FIP200 KI/KI MEFs were starved in HBSS for 2 h, fixed, and stained for endogenous LC3B. Representative images are shown. Bar, 10 μm. ( G ) Quantification of LC3B puncta number in starved MEF cells. At least 50 cells per experiment were counted. (**) P < 0.01. ( H ) Representative transmission electron microscopy images of starved MEF FIP200 +/+ and MEF FIP200 KI/KI cells. Arrows indicate autophagosomal structures ( left panel) and mitochondria ( right panel), respectively. Bars, 500 nm. ( I ) Genotypes of progeny from FIP200 +/ KI intercrosses.
    Figure Legend Snippet: Disruption of FIP200 interaction with Atg13 blocks autophagy in MEFs. ( A ) Schematic representation of the FIP200-4A mutant knock-in targeting vector and the targeted allele of the FIP200-4A mutant. Solid boxes represent regions of vector homology with the target locus; thick, numbered boxes represent the exons flanking homology regions. Site-directed mutagenesis was used to introduce the LQFL-to-AAAA mutation at the 582–585 amino acid residues. The asterisk denotes the mutated residues on exon 13. (EcoRV) EcoRV sites; (neo) neomycin-resistant gene cassette; (DTA) diphtheria toxin gene. P1 and P2 are a pair of primers for genotyping. P3 and P4 are a pair of primers for sequencing amplified genomic DNA with mutations. ( B ) Genomic DNA was extracted from mouse tails and analyzed by PCR using P1 and P2 to detect the FIP200 wild-type allele (178 base pairs [bp]) or knock-in allele (448 bp). Representative genotypes of a litter of mice from crosses of FIP200 +/ KI mice with wild-type B6 mice. ( C ) Representative genotypes of MEF cells prepared from embryonic day 13.5 (E13.5) embryos of intercrosses of FIP200 +/ KI mice. ( D ) Lysates from different MEFs were analyzed by immunoblotting using antibodies against various proteins as indicated. ( E ) MEFs were incubated in fresh growth medium (control) or HBSS (starvation) in the presence of 100 nM BafA1 for 2 h. Lysates were then prepared and analyzed by immunoblotting using antibodies against proteins as indicated. ( F ) FIP200 +/+ and FIP200 KI/KI MEFs were starved in HBSS for 2 h, fixed, and stained for endogenous LC3B. Representative images are shown. Bar, 10 μm. ( G ) Quantification of LC3B puncta number in starved MEF cells. At least 50 cells per experiment were counted. (**) P < 0.01. ( H ) Representative transmission electron microscopy images of starved MEF FIP200 +/+ and MEF FIP200 KI/KI cells. Arrows indicate autophagosomal structures ( left panel) and mitochondria ( right panel), respectively. Bars, 500 nm. ( I ) Genotypes of progeny from FIP200 +/ KI intercrosses.

    Techniques Used: Mutagenesis, Knock-In, Plasmid Preparation, Introduce, Sequencing, Amplification, Western Blot, Incubation, Staining, Transmission Assay, Electron Microscopy

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Cell Signaling Technology Inc phospho atg13 s355 d6j1w
    Phospho Atg13 S355 D6j1w, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho atg13 s355 d6j1w/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho atg13 s355 d6j1w - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc p atg13
    Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), <t>p-Atg13</t> (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    P Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p atg13 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc phosphor atg13 ser355
    Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, <t>ATG13,</t> ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).
    Phosphor Atg13 Ser355, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphor atg13 ser355/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphor atg13 ser355 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc anti atg13 s355
    (A) <t>ULK1-ATG13</t> shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.
    Anti Atg13 S355, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti atg13 s355/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti atg13 s355 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc rabbit anti phospho atg13
    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, <t>ATG13,</t> p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments
    Rabbit Anti Phospho Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho atg13 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc rabbit anti atg13 polyclonal antibody
    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, <t>ATG13,</t> p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments
    Rabbit Anti Atg13 Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti atg13 polyclonal antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti atg13 polyclonal antibody - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc rabbit anti phospho atg13 ser355 antibody
    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, <t>ATG13,</t> p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments
    Rabbit Anti Phospho Atg13 Ser355 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho atg13 ser355 antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho atg13 ser355 antibody - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    94
    Cell Signaling Technology Inc rabbit anti atg13
    Identification of residues 582–585 LQFL in FIP200, required for its interaction with <t>Atg13</t> and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .
    Rabbit Anti Atg13, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti atg13/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti atg13 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    Image Search Results


    Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

    doi: 10.1128/JVI.01537-21

    Figure Lengend Snippet: Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: Antibodies against Atg5 (2630), p-AKT (4060), AKT (4685), p-mTOR (5536), mTOR (2983), p-S6 (4858), S6 (2317), p-4E-BP1 (2855), 4E-BP1 (9644), p-AMPK (2535), AMPK (5831), p-TSC-2 (3614), TSC-2 (4308), p-Raptor (2083), Raptor (2280), p-Atg13 (26839), Atg13 (13273), ULK1 (8054), p-ULK1 (Ser757) (14202), p-ULK1 (Ser555) (5869), VPS15 (14580), VPS34 (4263), Beclin1 (3495), p-Beclin1 (84966), Atg14 (96752), Atg16L1 (8089), p62 (16177), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Infection, Western Blot, Quantitative RT-PCR, Transfection

    Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, ATG13, ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Journal: Cell Death & Disease

    Article Title: ω-6 Polyunsaturated fatty acids (linoleic acid) activate both autophagy and antioxidation in a synergistic feedback loop via TOR-dependent and TOR-independent signaling pathways

    doi: 10.1038/s41419-020-02750-0

    Figure Lengend Snippet: Serum-starved cells were treated with the indicated concentrations of LA or DHA for 24 h. The viability of the hepatocytes was evaluated with a CCK8 Assay Kit after LA treatment ( n = 3) ( a ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed ( n = 6) ( b ). Nuclear Nrf2 was examined by western blot analysis and quantitated after treatment with LA or DHA ( n = 3) ( c ). Representative confocal microscopic image of hepatocytes stained with AO. Scale bars, 50 μm ( d ). The presence of AO-stained intracellular autophagic vacuoles was demonstrated by flow cytometry, which revealed a dose-dependent increase in the red:green (FL2:FL1) fluorescence ratio, and the numbers of the AO-stained intracellular autophagic vacuoles were calculated ( e ). Representative confocal microscopic image of hepatocytes stained with MDC. Scale bars, 200 and 100 μm ( f ). The numbers of the MDC-stained intracellular autophagosomes were calculated with a fluorescence microplate assay of blue fluorescence intensity ( g ). Representative confocal microscopic image of hepatocytes stained with LysoTracker. Scale bars, 50 μm ( h ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, which revealed a dose-dependent increase in red (FL4) fluorescence intensity, and the numbers of the LysoTracker-stained intracellular autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity ( i ). The mRNA expression levels of key autophagy-related genes (Beclin1, ULK1, ATG101, ATG13, ATG5, ATG7, ATG12, ATG4b, LC3, GABARA, and P62) were analyzed ( n = 6) ( j ). The LC3-II/LC3-I ratio was examined by western blot analysis and quantitated after treatment with LA ( n = 3) ( k ). The results are presented as the mean with SEM and were analyzed using independent t- tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Article Snippet: Antibodies against AMPKα (cat. no. 5832), phosphor-AMPKα (Thr172) (cat. no. 50081), raptor (cat. no. 2280), phosphor- raptor (Ser792) (cat. no. 2083), TSC2 (cat. no. 4308), phosphor-TSC2 (Ser1387) (cat. no. 23402), ULK1 (cat. no. 8054), phosphor-ULK1 (Ser467) (cat. no. 4634), phosphor-ULK1 (Ser555) (cat. no. 5869), phosphor-ULK1 (Ser637) (cat. no. 14205), phosphor-ULK1 (Ser757) (cat. no. 14202), ATG13 (cat. no. 13468), phosphor-ATG13 (Ser355) (cat. no. 26839), TFEB (cat. no. 37785), and phosphor-TFEB (Ser211) (cat. no. 37681) were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: CCK-8 Assay, Expressing, Western Blot, Staining, Flow Cytometry, Fluorescence

    The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 μM MHY1485) ( n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptor Ser792 , T-TSC2, P-TSC2 Ser1387 , T-ULK1, P-ULK1 Ser467 , P-ULK1 Ser555 , P-ULK1 Ser637 , P-ULK1 Ser757 , T-ATG13, P-ATG13 Ser355 , T-TFEB, P-TFEB Ser211 , and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls ( a , b ). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 μm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group ( c , e ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator ( d , f ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator ( n = 6) ( g , h ). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Journal: Cell Death & Disease

    Article Title: ω-6 Polyunsaturated fatty acids (linoleic acid) activate both autophagy and antioxidation in a synergistic feedback loop via TOR-dependent and TOR-independent signaling pathways

    doi: 10.1038/s41419-020-02750-0

    Figure Lengend Snippet: The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 μM MHY1485) ( n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptor Ser792 , T-TSC2, P-TSC2 Ser1387 , T-ULK1, P-ULK1 Ser467 , P-ULK1 Ser555 , P-ULK1 Ser637 , P-ULK1 Ser757 , T-ATG13, P-ATG13 Ser355 , T-TFEB, P-TFEB Ser211 , and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls ( a , b ). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 μm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group ( c , e ). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator ( d , f ). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator ( n = 6) ( g , h ). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (* P ≥ 0.05).

    Article Snippet: Antibodies against AMPKα (cat. no. 5832), phosphor-AMPKα (Thr172) (cat. no. 50081), raptor (cat. no. 2280), phosphor- raptor (Ser792) (cat. no. 2083), TSC2 (cat. no. 4308), phosphor-TSC2 (Ser1387) (cat. no. 23402), ULK1 (cat. no. 8054), phosphor-ULK1 (Ser467) (cat. no. 4634), phosphor-ULK1 (Ser555) (cat. no. 5869), phosphor-ULK1 (Ser637) (cat. no. 14205), phosphor-ULK1 (Ser757) (cat. no. 14202), ATG13 (cat. no. 13468), phosphor-ATG13 (Ser355) (cat. no. 26839), TFEB (cat. no. 37785), and phosphor-TFEB (Ser211) (cat. no. 37681) were obtained from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Western Blot, Staining, Flow Cytometry, Fluorescence, Expressing

    (A) ULK1-ATG13 shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A) ULK1-ATG13 shows a mobility shift in mitosis. HeLa cells synchronized by double-thymidine release in the presence or absence of nocodazole were subjected to SDS-PAGE and western blot analysis. (B) ULK1-ATG13 undergoes band shift during mitotic progression. HeLa cells synchronized by double-thymidine and RO-3306 were released into mitosis for western blot analysis. (C) ULK1 is phosphorylated in nocodazole-arrested mitosis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine and nocodazole. The immunoprecipitates using the FLAG antibody were subjected to Coomassie Brilliant Blue R-250 staining and western blot analysis. Statistical analysis for relative serine/threonine phosphorylated ULK1 was shown in , lower panel. n = 4, ** p < 0.01. (D) The immunoprecipitates from were treated with or without λPP in the presence or absence of PPIs and then subjected to western blot analysis. (E) ULK1 undergoes mobility shift in both nocodazole- and STLC-arrested mitosis. HeLa cells synchronized with single-thymidine and nocodazole or STLC were analyzed by western blot analysis. The upper panel shows the immunoblotting and the lower panel shows the ratio of upshifted and nonshifted ULK1. One-way ANOVA followed by Tukey’s multiple comparison test was used for the analysis. n = 5, *** p < 0.001. (F) ULK1 undergoes phosphorylation-induced electrophoretic mobility shift in single-thymidine and nocodazole synchronized mitotic HCT 116 and RPE1 cells analyzed by western blot analysis. Numerical data underlying the figure panels are available in . Asyn, asynchronous; ATG, autophagy-related; DT-R2h, double-thymidine block and release for 2 hours; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; n.s., not significant; HCT 116, human colorectal cancer cells; PPI, phosphatase inhibitor; RPE1, human retinal pigmented epithelial cells; STLC, S-trityl-L-cysteine; ULK1, unc-51-like autophagy activating kinase 1; λPP, lambda phosphatase.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Mobility Shift, SDS Page, Western Blot, Electrophoretic Mobility Shift Assay, Staining, Blocking Assay, Immunoprecipitation

    (A-B) ULK1-ATG13 upshifted band in mitosis could be recognized by CDK substrate-specific antibodies. The 293T cells stably expressing FLAG-tagged mULK1 or ATG13 were synchronized by single-thymidine and released in the presence or absence of nocodazole. The coimmunoprecipitates and input were immunoblotted with specific antibodies. The phospho-signal was quantified relative to the FLAG antibody–detected bands (mULK1/ATG13-3FLAG) in IP; the IP signal of mULK1/ATG13-3FLAG was quantified relative to its input. Various cell cycle markers were detected to show the respective phases of cell cycle. ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200 kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; p-MAPK, phospho mitogen-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A-B) ULK1-ATG13 upshifted band in mitosis could be recognized by CDK substrate-specific antibodies. The 293T cells stably expressing FLAG-tagged mULK1 or ATG13 were synchronized by single-thymidine and released in the presence or absence of nocodazole. The coimmunoprecipitates and input were immunoblotted with specific antibodies. The phospho-signal was quantified relative to the FLAG antibody–detected bands (mULK1/ATG13-3FLAG) in IP; the IP signal of mULK1/ATG13-3FLAG was quantified relative to its input. Various cell cycle markers were detected to show the respective phases of cell cycle. ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200 kDa; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; p-MAPK, phospho mitogen-activated protein kinase; ULK1, unc-51-like autophagy activating kinase 1.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Stable Transfection, Expressing, Immunoprecipitation

    (A) Mitotic ATG13 undergoes mobility upshift in mitosis in electrophoresis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine in the presence or absence of nocodazole and coimmunoprecipitated by the FLAG antibody followed by western blot analysis. Size of endogenous ATG and expressed FLAG-tagged ATG13 were not distinguishable in electrophoresis here. (B) The effect of AMPK inhibition on ULK1 and ATG13 mobility shift in mitosis. The AMPK inhibitor Compound C was used to evaluate the role of AMPK on ULK1 or ATG13 mobility shift as . Phospho-AMPKα-T172 and phospho-Acetyl-CoA Carboxylase (Ser79) were used to indicate AMPK inhibition. (C) CDK1 inhibitor RO-3306 decreases the ATG13 band shift in mitosis. HeLa cells synchronized and treated as were subjected to western blot analysis. (D) ATG13 is phosphorylated and interacts with CDK1/cyclin B1 in mitosis. The 293T cells overexpressing FLAG-tagged ATG13 were synchronized by single-thymidine in the presence or absence of nocodazole. The coimmunoprecipitates by FLAG antibody were subjected to western blot analysis. (E-F) ATG13 is directly phosphorylated by purified CDK1/cyclin B complex in vitro. The ATG13 immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged ATG13 or ULK1 and purified CDK1/cyclin B complex were subjected to in vitro kinase assay with or without RO-3306 followed by western blot analysis. (E) shows representative western blot analysis of the ATG13 immunoprecipitates as substrate, and (F) shows the representative western blot analysis of the ULK1-WT coimmunoprecipitates as substrate. AMPK, AMP-activated protein kinase; ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A) Mitotic ATG13 undergoes mobility upshift in mitosis in electrophoresis. The 293T cells overexpressing FLAG-tagged mULK1 or GFP were synchronized by single-thymidine in the presence or absence of nocodazole and coimmunoprecipitated by the FLAG antibody followed by western blot analysis. Size of endogenous ATG and expressed FLAG-tagged ATG13 were not distinguishable in electrophoresis here. (B) The effect of AMPK inhibition on ULK1 and ATG13 mobility shift in mitosis. The AMPK inhibitor Compound C was used to evaluate the role of AMPK on ULK1 or ATG13 mobility shift as . Phospho-AMPKα-T172 and phospho-Acetyl-CoA Carboxylase (Ser79) were used to indicate AMPK inhibition. (C) CDK1 inhibitor RO-3306 decreases the ATG13 band shift in mitosis. HeLa cells synchronized and treated as were subjected to western blot analysis. (D) ATG13 is phosphorylated and interacts with CDK1/cyclin B1 in mitosis. The 293T cells overexpressing FLAG-tagged ATG13 were synchronized by single-thymidine in the presence or absence of nocodazole. The coimmunoprecipitates by FLAG antibody were subjected to western blot analysis. (E-F) ATG13 is directly phosphorylated by purified CDK1/cyclin B complex in vitro. The ATG13 immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged ATG13 or ULK1 and purified CDK1/cyclin B complex were subjected to in vitro kinase assay with or without RO-3306 followed by western blot analysis. (E) shows representative western blot analysis of the ATG13 immunoprecipitates as substrate, and (F) shows the representative western blot analysis of the ULK1-WT coimmunoprecipitates as substrate. AMPK, AMP-activated protein kinase; ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; IP, immunoprecipitation; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Electrophoresis, Western Blot, Inhibition, Mobility Shift, Electrophoretic Mobility Shift Assay, Purification, In Vitro, Kinase Assay, Immunoprecipitation

    (A) The mitotic-specific phosphorylated sites identified by MS in mitotic mULK1 compared with asynchronous mULK1. (B-C) S622/T635/T653 phosphorylation contributes to ULK1 mobility shift in mitosis. The 293T cells overexpressing FLAG-tagged mULK1-S622/T635/T653 mutants were synchronized with single-thymidine and nocodazole. Then immunoprecipitation for single mutant with FLAG antibody (B) or western blot analysis for double and triple mutant (C) was performed. (D-E) More sites contribute to ULK1 band shift. Based on triple 3A mutant, the other 8 Ser/Thr sites were mutated into Ala. The 293T cells expressing various mutants were synchronized into mitosis with thymidine and nocodazole for immunoprecipitation by FLAG antibody and western blot analysis. (F) ULK1-11A mutant was not upshifted or phosphorylated by CDK1/cyclin B kinase complex in vitro. The ULK1-11A mutant immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged mULK1 and purified CDK1/cyclin B complex were tested in an in vitro kinase assay and western blot analysis. (G) The phosphorylated sites identified by MS in mitotic ATG13 compared with asynchronous ATG13. (H-I) ATG13-T342/T332/S44/S224 phosphorylation contributes to ATG13 mobility shift in mitosis. HeLa-ATG13 KO cells (H) or 293T cells (I) overexpressing FLAG-tagged ATG13-T342/T332/S44/S224 mutants were synchronized with single-thymidine and nocodazole. Then western blot analysis for 4-site mutant (H) or immunoprecipitation and input for mutants with FLAG antibody (I) was performed. 3A, S622/T635/T653A; 5A, 3A-S479&S543A; 7A, 5A-S411&S413A; 9A, 5A-S413&T401&S403&S405A; 10A, 9A-T282A; 11A, 10A-T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; GFP, green fluorescent protein; IP, immunoprecipitation; MS, mass spectrometry; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A) The mitotic-specific phosphorylated sites identified by MS in mitotic mULK1 compared with asynchronous mULK1. (B-C) S622/T635/T653 phosphorylation contributes to ULK1 mobility shift in mitosis. The 293T cells overexpressing FLAG-tagged mULK1-S622/T635/T653 mutants were synchronized with single-thymidine and nocodazole. Then immunoprecipitation for single mutant with FLAG antibody (B) or western blot analysis for double and triple mutant (C) was performed. (D-E) More sites contribute to ULK1 band shift. Based on triple 3A mutant, the other 8 Ser/Thr sites were mutated into Ala. The 293T cells expressing various mutants were synchronized into mitosis with thymidine and nocodazole for immunoprecipitation by FLAG antibody and western blot analysis. (F) ULK1-11A mutant was not upshifted or phosphorylated by CDK1/cyclin B kinase complex in vitro. The ULK1-11A mutant immunoprecipitates from asynchronous 293T cells overexpressing FLAG-tagged mULK1 and purified CDK1/cyclin B complex were tested in an in vitro kinase assay and western blot analysis. (G) The phosphorylated sites identified by MS in mitotic ATG13 compared with asynchronous ATG13. (H-I) ATG13-T342/T332/S44/S224 phosphorylation contributes to ATG13 mobility shift in mitosis. HeLa-ATG13 KO cells (H) or 293T cells (I) overexpressing FLAG-tagged ATG13-T342/T332/S44/S224 mutants were synchronized with single-thymidine and nocodazole. Then western blot analysis for 4-site mutant (H) or immunoprecipitation and input for mutants with FLAG antibody (I) was performed. 3A, S622/T635/T653A; 5A, 3A-S479&S543A; 7A, 5A-S411&S413A; 9A, 5A-S413&T401&S403&S405A; 10A, 9A-T282A; 11A, 10A-T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; GFP, green fluorescent protein; IP, immunoprecipitation; MS, mass spectrometry; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Mobility Shift, Immunoprecipitation, Mutagenesis, Western Blot, Electrophoretic Mobility Shift Assay, Expressing, In Vitro, Purification, Kinase Assay, Mass Spectrometry

    (A) The localization of mutant ULK1-11A and ATG13-4A in mitosis. Cells released from thymidine block for 10 hours were fixed with 3.7% formaldehyde for 20 minutes at room temperature and then for blocking and FLAG antibody, Alexa-488 conjugated secondary antibody, DAPI staining. Scale bar, 10 μm. (B) The ULK1 kinase activity is not affected by the 11A mutation. ATG13-Ser355 is the substrate of ULK1, which corresponds to Ser318 of ATG13 isoform 2. ULK1-KO 293T cells reconstituted with ULK1-WT, ULK1-11A, or ULK1-K46I were plated and lysed with M-PER. The western blot for ATG13-Ser355 indicated that ULK1 activity was not affected by 11A mutation. n = 3. (C) Autophagy activity was examined in ULK1&ATG13 double WT or mutant (“Mut”) cells expressing GFP-LC3-RFP by flow cytometry. ULK1&ATG13 double WT or mutant cells, HeLa, or HeLa- ULK1&ATG13 DKO cells stably expressing GFP-LC3-RFP were established by infection of retrovirus packaged by pMRX-IP-GFP-LC3-RFP and helper plasmids Vsvg and pMLV. Mitotic cells were collected by shake-off from thymidine and nocodazole synchronized cells for flow cytometry detection. The GFP/RFP ratio inversely correlated with autophagy activity. n = 4, *** p < 0.001, **** p < 0.0001. (D) The autophagic flux of HeLa-DKO cells stably overexpressing double WT or mutant FLAG-tagged mULK1 and ATG13. Cells synchronized to mitosis with thymidine and nocodazole were shaken off and treated with or without autophagy inhibitor 25 μM CQ for 1 hour. The left panel is a representative western blot, and the right panel is the statistical result for LC3B-II and p62. n = 3, * p < 0.05, ** p < 0.01. (E) LC3B puncta number was counted, and the micrographs were captured by Zeiss LSM710 confocal microscope. Scale bar, 10 μm. n = 43, ** p < 0.01. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CQ, chloroquine; Ctrl, control; DAPI, 4 DAPdiamidino-2-phenylindole; DKO, double knockout; GFP, green fluorescent protein; KO, knockout; M-PER, Mammalian Protein Extraction Reagent; mULK1, mouse ULK1; n.s., not significant; RFP, red fluorescent protein; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A) The localization of mutant ULK1-11A and ATG13-4A in mitosis. Cells released from thymidine block for 10 hours were fixed with 3.7% formaldehyde for 20 minutes at room temperature and then for blocking and FLAG antibody, Alexa-488 conjugated secondary antibody, DAPI staining. Scale bar, 10 μm. (B) The ULK1 kinase activity is not affected by the 11A mutation. ATG13-Ser355 is the substrate of ULK1, which corresponds to Ser318 of ATG13 isoform 2. ULK1-KO 293T cells reconstituted with ULK1-WT, ULK1-11A, or ULK1-K46I were plated and lysed with M-PER. The western blot for ATG13-Ser355 indicated that ULK1 activity was not affected by 11A mutation. n = 3. (C) Autophagy activity was examined in ULK1&ATG13 double WT or mutant (“Mut”) cells expressing GFP-LC3-RFP by flow cytometry. ULK1&ATG13 double WT or mutant cells, HeLa, or HeLa- ULK1&ATG13 DKO cells stably expressing GFP-LC3-RFP were established by infection of retrovirus packaged by pMRX-IP-GFP-LC3-RFP and helper plasmids Vsvg and pMLV. Mitotic cells were collected by shake-off from thymidine and nocodazole synchronized cells for flow cytometry detection. The GFP/RFP ratio inversely correlated with autophagy activity. n = 4, *** p < 0.001, **** p < 0.0001. (D) The autophagic flux of HeLa-DKO cells stably overexpressing double WT or mutant FLAG-tagged mULK1 and ATG13. Cells synchronized to mitosis with thymidine and nocodazole were shaken off and treated with or without autophagy inhibitor 25 μM CQ for 1 hour. The left panel is a representative western blot, and the right panel is the statistical result for LC3B-II and p62. n = 3, * p < 0.05, ** p < 0.01. (E) LC3B puncta number was counted, and the micrographs were captured by Zeiss LSM710 confocal microscope. Scale bar, 10 μm. n = 43, ** p < 0.01. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CQ, chloroquine; Ctrl, control; DAPI, 4 DAPdiamidino-2-phenylindole; DKO, double knockout; GFP, green fluorescent protein; KO, knockout; M-PER, Mammalian Protein Extraction Reagent; mULK1, mouse ULK1; n.s., not significant; RFP, red fluorescent protein; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Mutagenesis, Blocking Assay, Staining, Activity Assay, Western Blot, Expressing, Flow Cytometry, Stable Transfection, Infection, Microscopy, Double Knockout, Knock-Out, Protein Extraction

    (A) ULK1-ATG13 DKO inhibits S/G2 and G2/M transitions. HeLa WT or ULK1&ATG13-DKO cells synchronized into mitosis were subjected to PI and pH3(S10) co-staining for cell cycle and mitotic index analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (B) Representative western blot analysis suggests that ULK1&ATG13 DKO inhibits mitotic entry, which is shown by mitotic markers and CDK1 substrate phosphorylation. (C) Mitotic exit of ULK1, ATG13, or ULK1&ATG13 KO cells. Cells were synchronized into mitosis with thymidine and NOC and released into NOC-free complete DMEM medium for different timepoints and then subjected to either PI and pH3(S10) co-staining or cyclin B1 staining for cell cycle, mitotic index, and cyclin B1 level analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (D) ULK1&ATG13 DKO inhibits cell proliferation. HeLa WT or ULK1&ATG13 DKO cells were plated at 1 × 10 5 cells/mL and cultured for 1, 2, or 3 days. The cell number was counted by flow cytometry and the doubling time was calculated. T D indicates the average cell doubling time and is calculated as: T D = t * [lg2/(lgNt − lgN0)], where t is the culture time, Nt is the cell number after culturing, and N0 is the original cell number plated. n = 3, * p < 0.05, *** p < 0.001. (E-G) The role of ULK1-ATG13 phosphorylation in cell cycle progression and cell proliferation. ULK1&ATG13-DKO cells reconstituted with WT mULK1-3FLAG and ATG13-3FLAG or mutant (“Mut”) mULK1-11A and ATG13-4A, HeLa, or ULK1&ATG13-DKO cells were treated as (A)/(B) and (D). The mitotic exit (G1%) is the percentage of cells in G1 phase when released for indicated time. n = 3, ** p < 0.01, *** p < 0.001. (H) The relative tumor volume growth curve of nude mice bearing different tumors with or without SBI-0206965. The nude mice were injected with cells (1 × 10 7 ) in 100 μL PBS/Matrigel Matrix (1:1). Seven days postimplantation, 5 mice in each group were injected with 0.5% (M/V) methyl cellulose or SBI-0206965 in 0.5% methyl cellulose (20 mg/kg/d) every day for 33 days. Tumor growth was evaluated every day and tumor volume was calculated as: volume = 1/2 (length × width 2 ). (I) Tumor growth in nude mice bearing WT or KO cells with or without ULK1 kinase inhibitor SBI-0206965. The protocols were indicated as and the mice were sacrificed, and tumors were harvested and weighed up at the end of the experiment. n = 5, * p < 0.05, *** p < 0.001. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; DKO, double knockout; DMEM, Dulbecco’s Modified Eagle Medium; KO, knockout; mULK1, mouse ULK1; Myt1, myelin transcription factor 1; NOC, nocodazole; n.s., not significant; pH3(S10), phospho histone H3 serine 10; PI, propidium iodide; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Journal: PLoS Biology

    Article Title: ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle

    doi: 10.1371/journal.pbio.3000288

    Figure Lengend Snippet: (A) ULK1-ATG13 DKO inhibits S/G2 and G2/M transitions. HeLa WT or ULK1&ATG13-DKO cells synchronized into mitosis were subjected to PI and pH3(S10) co-staining for cell cycle and mitotic index analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (B) Representative western blot analysis suggests that ULK1&ATG13 DKO inhibits mitotic entry, which is shown by mitotic markers and CDK1 substrate phosphorylation. (C) Mitotic exit of ULK1, ATG13, or ULK1&ATG13 KO cells. Cells were synchronized into mitosis with thymidine and NOC and released into NOC-free complete DMEM medium for different timepoints and then subjected to either PI and pH3(S10) co-staining or cyclin B1 staining for cell cycle, mitotic index, and cyclin B1 level analysis by flow cytometry. n = 3, * p < 0.05, ** p < 0.01. (D) ULK1&ATG13 DKO inhibits cell proliferation. HeLa WT or ULK1&ATG13 DKO cells were plated at 1 × 10 5 cells/mL and cultured for 1, 2, or 3 days. The cell number was counted by flow cytometry and the doubling time was calculated. T D indicates the average cell doubling time and is calculated as: T D = t * [lg2/(lgNt − lgN0)], where t is the culture time, Nt is the cell number after culturing, and N0 is the original cell number plated. n = 3, * p < 0.05, *** p < 0.001. (E-G) The role of ULK1-ATG13 phosphorylation in cell cycle progression and cell proliferation. ULK1&ATG13-DKO cells reconstituted with WT mULK1-3FLAG and ATG13-3FLAG or mutant (“Mut”) mULK1-11A and ATG13-4A, HeLa, or ULK1&ATG13-DKO cells were treated as (A)/(B) and (D). The mitotic exit (G1%) is the percentage of cells in G1 phase when released for indicated time. n = 3, ** p < 0.01, *** p < 0.001. (H) The relative tumor volume growth curve of nude mice bearing different tumors with or without SBI-0206965. The nude mice were injected with cells (1 × 10 7 ) in 100 μL PBS/Matrigel Matrix (1:1). Seven days postimplantation, 5 mice in each group were injected with 0.5% (M/V) methyl cellulose or SBI-0206965 in 0.5% methyl cellulose (20 mg/kg/d) every day for 33 days. Tumor growth was evaluated every day and tumor volume was calculated as: volume = 1/2 (length × width 2 ). (I) Tumor growth in nude mice bearing WT or KO cells with or without ULK1 kinase inhibitor SBI-0206965. The protocols were indicated as and the mice were sacrificed, and tumors were harvested and weighed up at the end of the experiment. n = 5, * p < 0.05, *** p < 0.001. Numerical data underlying the figure panels are available in . 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CDK, cyclin-dependent kinase; DKO, double knockout; DMEM, Dulbecco’s Modified Eagle Medium; KO, knockout; mULK1, mouse ULK1; Myt1, myelin transcription factor 1; NOC, nocodazole; n.s., not significant; pH3(S10), phospho histone H3 serine 10; PI, propidium iodide; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.

    Article Snippet: The autophagy antibody sampler kit (#4445), ULK1 Antibody Sampler Kit (#8359), Autophagy Induction (ULK1 Complex) Antibody Sampler Kit (#46486), the cell cycle regulation antibody sampler kit II (#9870), Phospho-(Ser) Kinase Substrate Antibody Sampler Kit (#9615), Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) (#9391), anti-ULK1 (#4776) antibody, AMPK and ACC Antibody Sampler Kit (#9957), anti-p62 (#5114) antibody, anti-ATG13(S355) (#26839) antibody, anti-ATG7 (#8558) antibody, anti-ATG9A (#13509) antibody, the HRP-linked anti-rabbit, and anti-mouse IgG antibodies were all from Cell Signaling Technology.

    Techniques: Staining, Flow Cytometry, Western Blot, Cell Culture, Mutagenesis, Injection, Double Knockout, Modification, Knock-Out

    a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments

    Journal: Cell Death & Disease

    Article Title: TOPK inhibits autophagy by phosphorylating ULK1 and promotes glioma resistance to TMZ

    doi: 10.1038/s41419-019-1805-9

    Figure Lengend Snippet: a TOPK-overexpression-U118MG cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were harvested at 80% confluence. ULK1, ATG13, p-ATG13, Beclin-1, p-Beclin-1, TOPK, and Tubulin were analyzed using western blots. c Hs683 (left) and H4 (right) cells were treated with different concentrations of OTS964 for 48 h, and the samples were resolved by SDS–PAGE and analyzed by western blots using the indicated antibodies. d TOPK-silencing-Hs683 (left) and TOPK-silencing-H4 (right) cells were treated with EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. e Hs683 (left) and -H4 (right) cells were treated with or without OTS964 (1 μM) for 48 h, then cells were exposed to EGF (20 ng/ml) for 15 min followed by the addition of CHX (100 μg/ml) for 6 and 8 h before extraction. ULK1 and Tubulin were analyzed using western blots. f H4 cells were treated with or without OTS964 (1 μM) for 48 h, and then cells were exposed to CHX (100 μg/ml) for 6 and 8 h before extraction. TOPK and Tubulin were analyzed using western blots. Data represent the results of triplicate experiments

    Article Snippet: The following primary antibodies were used: rabbit anti-P62/SQSTM1 (1:1000, Proteintech Group, 18420-1-AP), mouse anti-TOPK (1:1000, Santa Cruz, sc-293028), rabbit anti-LC3B (1:1000, Cell Signaling Technology, 2775), rabbit anti-Caspase-3 (1:1000, Cell Signaling Technology, #9662), rabbit anti-Cleaved-Caspase-3 (1:500, Cell Signaling Technology, #9664), rabbit anti-ULK1 (1:500, Cell Signaling Technology, 8054), rabbit anti-ATG13 (1:1000, Cell Signaling Technology, 13468), rabbit anti-phospho-ATG13 (1:500, Cell Signaling Technology, 26839), rabbit anti-Histone H3 (1:1000, Cell Signaling Technology, 4499), rabbit anti-phospho-Histone H3 (1:1000, Cell Signaling Technology, 3377S), rabbit anti-HA (1:1000, Cell Signaling Technology, 3724), mouse anti-HA (1:200, Pregene, PRE003F), mouse anti-Alpha Tubulin (1:1000, Proteintech Group, 66031-1-Ig), mouse anti-His (1:1000, Pregene, PRE004F), rabbit anti-Flag (1:1000, Sigma-Aldrich, F7425).

    Techniques: Over Expression, Western Blot, SDS Page

    a PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. c PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Then cells were treated with EGF (20 ng/ml) for 15 min, followed by the addition of CHX (100 μg/ml) for 6, 8, and 12 h before extraction. HA and Tubulin were analyzed using western blots (left). Data of c represent the results of triplicate experiments. The densities of HA/Tubulin (right) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. d HA-ULK1-WT or HA-ULK1-AAA, Flag-ubiquitin, and GAL4-TOPK were cotransfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-Flag, anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. e PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Cells were harvested 48 h later. LC3B, HA, and Tubulin were analyzed using western blots. f PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. 24 h later, cells were starved for 24 h, then rapamycin (50 nM) and CQ (50 μM) were added, respectively, for 15 min and 1 h. LC3B, HA, and Tubulin were analyzed using western blots. The densities of LC3-II/Tubulin (Fig. 6 f , bottom) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. Data represent the results of triplicate experiments

    Journal: Cell Death & Disease

    Article Title: TOPK inhibits autophagy by phosphorylating ULK1 and promotes glioma resistance to TMZ

    doi: 10.1038/s41419-019-1805-9

    Figure Lengend Snippet: a PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. ATG13, p-ATG13, Beclin-1, p-Beclin-1, HA, and Tubulin were analyzed using western blots. b PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. c PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Then cells were treated with EGF (20 ng/ml) for 15 min, followed by the addition of CHX (100 μg/ml) for 6, 8, and 12 h before extraction. HA and Tubulin were analyzed using western blots (left). Data of c represent the results of triplicate experiments. The densities of HA/Tubulin (right) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. d HA-ULK1-WT or HA-ULK1-AAA, Flag-ubiquitin, and GAL4-TOPK were cotransfected into HEK293T cells, 48 h later, cells were extracted, immunoprecipitated with anti-HA, and probed with anti-Flag, anti-FIP200, anti-ATG101, and anti-ATG13 by western blots. Equal protein loading and transfection efficiency were determined by western blots using the total extracts. e PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. Cells were harvested 48 h later. LC3B, HA, and Tubulin were analyzed using western blots. f PRK5-HA-ULK1-WT and PRK5-HA-ULK1-Ser469/495/533AAA were transiently transfected into HEK293T cells as indicated. 24 h later, cells were starved for 24 h, then rapamycin (50 nM) and CQ (50 μM) were added, respectively, for 15 min and 1 h. LC3B, HA, and Tubulin were analyzed using western blots. The densities of LC3-II/Tubulin (Fig. 6 f , bottom) were determined by Image J. The data were presented in the form of mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001. Data represent the results of triplicate experiments

    Article Snippet: The following primary antibodies were used: rabbit anti-P62/SQSTM1 (1:1000, Proteintech Group, 18420-1-AP), mouse anti-TOPK (1:1000, Santa Cruz, sc-293028), rabbit anti-LC3B (1:1000, Cell Signaling Technology, 2775), rabbit anti-Caspase-3 (1:1000, Cell Signaling Technology, #9662), rabbit anti-Cleaved-Caspase-3 (1:500, Cell Signaling Technology, #9664), rabbit anti-ULK1 (1:500, Cell Signaling Technology, 8054), rabbit anti-ATG13 (1:1000, Cell Signaling Technology, 13468), rabbit anti-phospho-ATG13 (1:500, Cell Signaling Technology, 26839), rabbit anti-Histone H3 (1:1000, Cell Signaling Technology, 4499), rabbit anti-phospho-Histone H3 (1:1000, Cell Signaling Technology, 3377S), rabbit anti-HA (1:1000, Cell Signaling Technology, 3724), mouse anti-HA (1:200, Pregene, PRE003F), mouse anti-Alpha Tubulin (1:1000, Proteintech Group, 66031-1-Ig), mouse anti-His (1:1000, Pregene, PRE004F), rabbit anti-Flag (1:1000, Sigma-Aldrich, F7425).

    Techniques: Transfection, Western Blot, Immunoprecipitation

    Identification of residues 582–585 LQFL in FIP200, required for its interaction with Atg13 and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .

    Journal: Genes & Development

    Article Title: Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    doi: 10.1101/gad.276428.115

    Figure Lengend Snippet: Identification of residues 582–585 LQFL in FIP200, required for its interaction with Atg13 and autophagy induction. ( A ) Schematic of the FIP200 fragments used in B – E . A white box marks residues 573–592, required for Atg13 binding. ( B – D ) HEK293T cells were cotransfected with expression vectors encoding Flag-tagged Atg13 and HA-tagged FIP200 or its segments or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA agarose beads followed by immunoblotting with antibodies as indicated. ( E ) The GFP-tagged FIP200 N-terminal or C-terminal segments indicated in A were transfected into HEK293T cells. The lysates were prepared and immunoprecipitated by anti-GFP antibody followed by immunoblotting with antibodies as indicated. ( F ) HEK293T cells were transfected with expression vectors encoding GFP-tagged-FIP200 or its mutants as indicated. Lysates were prepared and immunoprecipitated by anti-GFP followed by immunoblotting with antibodies as indicated. ( G – I ) GFP-tagged FIP200 or FIP200-4A mutant was cotransfected with Myc-FAK ( G ), Myc-Tsc1 ( H ), or Myc-P53 ( I ), respectively, into HEK293T cells. Lysates were prepared and immunoprecipitated with anti-GFP antibody followed by immunoblotting using antibodies as indicated. Please note that the bands marked by asterisks in the bottom panels of B and D are Flag-tagged Atg13 that was not stripped after immunoblotting using anti-Flag. Molecular weight markers (kilodaltons) are shown at the right .

    Article Snippet: The following antibodies were used: monoclonal anti-HA-agarose (Sigma, A2095), rabbit anti-HA (Santa Cruz Biotechnology, sc-805), rabbit anti-Flag (SAB) (Signalwayantibody, T503), rabbit anti-GFP (Invitrogen, A11222), mouse anti-Myc (Santa Cruz Biotechnology, sc-40), rabbit anti-ULK1 (Cell Signaling, 8054), rabbit anti-Atg13 (Cell Signaling, 6940), rabbit anti-FIP200 (Ptglab, 17250), rabbit anti-LC3B (Cell Signaling, 2775), rabbit anti-P62 (Cell Signaling, 5114), rabbit anti-Ser240/244 phospho-S6 (Cell Signaling, 5364), rabbit anti-cleaved caspase 3 (Cell Signaling, 9661), rabbit anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling,4668), and rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033).

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Molecular Weight

    The FIP200-4A mutant unable to bind ATG13 is defective in amino acid starvation-induced autophagy. ( A ) HEK293T cells were transfected with expression vectors encoding HA-tagged FIP200 and FIP200-4A mutant or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA followed by immunoblotting using anti-HA ( top ), anti-ULK1 ( middle ), or anti-Atg13 ( bottom ). ( B ) GFP-FIP200 ( left ) and GFP-FIP200-4A mutant puncta formation in starved HEK293T cells. ( C , D ) shRNA FIP200 knockdown HEK293T cells reconstituted with shRNA-resistant GFP-FIP200 or the GFP-FIP200-4A mutant were either left untreated or starved (Hank's balanced salt solution [HBSS]) for 2 h and then analyzed by immunoblotting with the indicated antibodies. ( C ) BafA1 (100 nM), an inhibitor of lysosome degradation, was included for 2 h in samples as indicated. ( D ) Quantification of LC3I-to-LC3II conversion in FIP200 knockdown HEK293T cells reconstituted with GFP-FIP200 and GFP-FIP200-4A upon starvation as shown in C . (**) P < 0.01.

    Journal: Genes & Development

    Article Title: Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    doi: 10.1101/gad.276428.115

    Figure Lengend Snippet: The FIP200-4A mutant unable to bind ATG13 is defective in amino acid starvation-induced autophagy. ( A ) HEK293T cells were transfected with expression vectors encoding HA-tagged FIP200 and FIP200-4A mutant or vector alone as a control as indicated. Lysates were prepared and immunoprecipitated by anti-HA followed by immunoblotting using anti-HA ( top ), anti-ULK1 ( middle ), or anti-Atg13 ( bottom ). ( B ) GFP-FIP200 ( left ) and GFP-FIP200-4A mutant puncta formation in starved HEK293T cells. ( C , D ) shRNA FIP200 knockdown HEK293T cells reconstituted with shRNA-resistant GFP-FIP200 or the GFP-FIP200-4A mutant were either left untreated or starved (Hank's balanced salt solution [HBSS]) for 2 h and then analyzed by immunoblotting with the indicated antibodies. ( C ) BafA1 (100 nM), an inhibitor of lysosome degradation, was included for 2 h in samples as indicated. ( D ) Quantification of LC3I-to-LC3II conversion in FIP200 knockdown HEK293T cells reconstituted with GFP-FIP200 and GFP-FIP200-4A upon starvation as shown in C . (**) P < 0.01.

    Article Snippet: The following antibodies were used: monoclonal anti-HA-agarose (Sigma, A2095), rabbit anti-HA (Santa Cruz Biotechnology, sc-805), rabbit anti-Flag (SAB) (Signalwayantibody, T503), rabbit anti-GFP (Invitrogen, A11222), mouse anti-Myc (Santa Cruz Biotechnology, sc-40), rabbit anti-ULK1 (Cell Signaling, 8054), rabbit anti-Atg13 (Cell Signaling, 6940), rabbit anti-FIP200 (Ptglab, 17250), rabbit anti-LC3B (Cell Signaling, 2775), rabbit anti-P62 (Cell Signaling, 5114), rabbit anti-Ser240/244 phospho-S6 (Cell Signaling, 5364), rabbit anti-cleaved caspase 3 (Cell Signaling, 9661), rabbit anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling,4668), and rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033).

    Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, shRNA

    Disruption of FIP200 interaction with Atg13 blocks autophagy in MEFs. ( A ) Schematic representation of the FIP200-4A mutant knock-in targeting vector and the targeted allele of the FIP200-4A mutant. Solid boxes represent regions of vector homology with the target locus; thick, numbered boxes represent the exons flanking homology regions. Site-directed mutagenesis was used to introduce the LQFL-to-AAAA mutation at the 582–585 amino acid residues. The asterisk denotes the mutated residues on exon 13. (EcoRV) EcoRV sites; (neo) neomycin-resistant gene cassette; (DTA) diphtheria toxin gene. P1 and P2 are a pair of primers for genotyping. P3 and P4 are a pair of primers for sequencing amplified genomic DNA with mutations. ( B ) Genomic DNA was extracted from mouse tails and analyzed by PCR using P1 and P2 to detect the FIP200 wild-type allele (178 base pairs [bp]) or knock-in allele (448 bp). Representative genotypes of a litter of mice from crosses of FIP200 +/ KI mice with wild-type B6 mice. ( C ) Representative genotypes of MEF cells prepared from embryonic day 13.5 (E13.5) embryos of intercrosses of FIP200 +/ KI mice. ( D ) Lysates from different MEFs were analyzed by immunoblotting using antibodies against various proteins as indicated. ( E ) MEFs were incubated in fresh growth medium (control) or HBSS (starvation) in the presence of 100 nM BafA1 for 2 h. Lysates were then prepared and analyzed by immunoblotting using antibodies against proteins as indicated. ( F ) FIP200 +/+ and FIP200 KI/KI MEFs were starved in HBSS for 2 h, fixed, and stained for endogenous LC3B. Representative images are shown. Bar, 10 μm. ( G ) Quantification of LC3B puncta number in starved MEF cells. At least 50 cells per experiment were counted. (**) P < 0.01. ( H ) Representative transmission electron microscopy images of starved MEF FIP200 +/+ and MEF FIP200 KI/KI cells. Arrows indicate autophagosomal structures ( left panel) and mitochondria ( right panel), respectively. Bars, 500 nm. ( I ) Genotypes of progeny from FIP200 +/ KI intercrosses.

    Journal: Genes & Development

    Article Title: Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

    doi: 10.1101/gad.276428.115

    Figure Lengend Snippet: Disruption of FIP200 interaction with Atg13 blocks autophagy in MEFs. ( A ) Schematic representation of the FIP200-4A mutant knock-in targeting vector and the targeted allele of the FIP200-4A mutant. Solid boxes represent regions of vector homology with the target locus; thick, numbered boxes represent the exons flanking homology regions. Site-directed mutagenesis was used to introduce the LQFL-to-AAAA mutation at the 582–585 amino acid residues. The asterisk denotes the mutated residues on exon 13. (EcoRV) EcoRV sites; (neo) neomycin-resistant gene cassette; (DTA) diphtheria toxin gene. P1 and P2 are a pair of primers for genotyping. P3 and P4 are a pair of primers for sequencing amplified genomic DNA with mutations. ( B ) Genomic DNA was extracted from mouse tails and analyzed by PCR using P1 and P2 to detect the FIP200 wild-type allele (178 base pairs [bp]) or knock-in allele (448 bp). Representative genotypes of a litter of mice from crosses of FIP200 +/ KI mice with wild-type B6 mice. ( C ) Representative genotypes of MEF cells prepared from embryonic day 13.5 (E13.5) embryos of intercrosses of FIP200 +/ KI mice. ( D ) Lysates from different MEFs were analyzed by immunoblotting using antibodies against various proteins as indicated. ( E ) MEFs were incubated in fresh growth medium (control) or HBSS (starvation) in the presence of 100 nM BafA1 for 2 h. Lysates were then prepared and analyzed by immunoblotting using antibodies against proteins as indicated. ( F ) FIP200 +/+ and FIP200 KI/KI MEFs were starved in HBSS for 2 h, fixed, and stained for endogenous LC3B. Representative images are shown. Bar, 10 μm. ( G ) Quantification of LC3B puncta number in starved MEF cells. At least 50 cells per experiment were counted. (**) P < 0.01. ( H ) Representative transmission electron microscopy images of starved MEF FIP200 +/+ and MEF FIP200 KI/KI cells. Arrows indicate autophagosomal structures ( left panel) and mitochondria ( right panel), respectively. Bars, 500 nm. ( I ) Genotypes of progeny from FIP200 +/ KI intercrosses.

    Article Snippet: The following antibodies were used: monoclonal anti-HA-agarose (Sigma, A2095), rabbit anti-HA (Santa Cruz Biotechnology, sc-805), rabbit anti-Flag (SAB) (Signalwayantibody, T503), rabbit anti-GFP (Invitrogen, A11222), mouse anti-Myc (Santa Cruz Biotechnology, sc-40), rabbit anti-ULK1 (Cell Signaling, 8054), rabbit anti-Atg13 (Cell Signaling, 6940), rabbit anti-FIP200 (Ptglab, 17250), rabbit anti-LC3B (Cell Signaling, 2775), rabbit anti-P62 (Cell Signaling, 5114), rabbit anti-Ser240/244 phospho-S6 (Cell Signaling, 5364), rabbit anti-cleaved caspase 3 (Cell Signaling, 9661), rabbit anti-phospho-JNK (Thr183/Tyr185) (Cell Signaling,4668), and rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033).

    Techniques: Mutagenesis, Knock-In, Plasmid Preparation, Introduce, Sequencing, Amplification, Western Blot, Incubation, Staining, Transmission Assay, Electron Microscopy