ptdins3p  (Echelon Biosciences)


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    Echelon Biosciences ptdins3p
    Ptdins3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptdins3p  (Echelon Biosciences)


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    Echelon Biosciences ptdins3p
    Ptdins3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 3 p  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 p
    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) <t>PI(3)P</t> levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
    Anti Pi 3 P, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases"

    Article Title: Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.709398

    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) PI(3)P levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
    Figure Legend Snippet: TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) PI(3)P levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .

    Techniques Used: Activity Assay, Quantitation Assay, Cell Culture, Activation Assay

    Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .
    Figure Legend Snippet: Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .

    Techniques Used: Fluorescence, Microscopy, Quantitation Assay

    anti gst  (Echelon Biosciences)


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    Echelon Biosciences anti gst
    Anti Gst, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p40px domain  (Echelon Biosciences)


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    Echelon Biosciences p40px domain
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    recombinant glutathione s transferase gst  (Echelon Biosciences)


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    Echelon Biosciences recombinant glutathione s transferase gst
    Recombinant Glutathione S Transferase Gst, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptdins3p  (Echelon Biosciences)


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    Echelon Biosciences ptdins3p
    Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of <t>PtdIns3P</t> was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.
    Ptdins3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14"

    Article Title: The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1140293

    Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of PtdIns3P was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.
    Figure Legend Snippet: Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of PtdIns3P was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.

    Techniques Used: Activation Assay, Activity Assay, Incubation, Dot Blot, In Vitro, Isolation, Cell Culture, Plasmid Preparation, Fluorescence, Microscopy, Staining

    ptdins3p  (Echelon Biosciences)


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    Echelon Biosciences ptdins3p
    Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of <t>PtdIns3P</t> was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.
    Ptdins3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14"

    Article Title: The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1140293

    Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of PtdIns3P was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.
    Figure Legend Snippet: Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of PtdIns3P was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P < 0.01 vs. full medium). N.D. means “not detected.” (C) The kinase activity of ATG14-associated PIK3C3 is enhanced by Torin1, rapamycin or starvation. HEK293T cells were incubated in DMEM or EBSS or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. The in vitro reaction was conducted as described in (A). Wortmannin (100 nM) was added during the reaction. (D) Quantitative analysis of PtdIns3P production in (C). Values are mean ± SD (*P < 0.05 and **P < 0.01 versus full medium). (E) ATG13 is important for starvation-induced activation of the ATG14-containing PtdIns3K complex. ATG14 immunoprecipitates were isolated from the indicated cells cultured in either DMEM or EBSS for 1 h. The kinase activity of the immunoprecipitates was analyzed as described in (A). (F) Quantitative analysis of PtdIns3P production in (E). (G) ATG14 Ser29 phosphorylation is important for the kinase activity of ATG14-associated PIK3C3. ATG14 immunoprecipitates were obtained from WT ATG14-, ATG14S29A-, or ATG14S29D-reconstituted HCT116 cells. The in vitro reaction was conducted as described in (A). (H) Quantitative analysis of the results from (G). Values are mean ± SD (*P < 0.05; **P < 0.01). (I) ATG14 Ser29 phosphorylation is important for ATG14 puncta formation and their colocalization with 2xFYVE puncta. HCT116 cells reconstituted with WT ATG14, ATG14S29A, or ATG14S29D were transiently transduced by a plasmid encoding RFP-2xFYVE. The cells were incubated in either DMEM or EBSS for 1 h. Endogenous ATG14 was immunostained using anti-ATG14 antibody, and visualized together with RFP-2xFYVE by fluorescence microscope. Nuclei were stained by DAPI (blue). Scale bar: 10 μm. (J to L) Quantitative analysis of the results from (I) (*, P < 0.05; **, P < 0.01; ****, P < 0.0001, n≥35 , the Student t test). Mean and SEM are shown as horizontal bars.

    Techniques Used: Activation Assay, Activity Assay, Incubation, Dot Blot, In Vitro, Isolation, Cell Culture, Plasmid Preparation, Fluorescence, Microscopy, Staining

    anti pi 3 p  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences anti pi 3 p
    A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of <t>PI(3)P</t> and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.
    Anti Pi 3 P, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex"

    Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004626

    A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of PI(3)P and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.
    Figure Legend Snippet: A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of PI(3)P and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.

    Techniques Used: Generated, Transgenic Assay, Staining, Incubation

    A . Table showing results from PI(3)P ELISA. Average 450 nm absorbance, p-value of comparison to Becn1 deficient MEFs. PI(3)P concentration is determined by comparison to a standard curve. Quantification of 450 nm absorbance of control, Becn1 deficient, and Becn1 revertant MEFs (6,5,6 replicates); statistics using a one-tailed t-test p = 0.0032, 0.0336. B . Anti-beclin 1, and -actin blots of MEF cell lysates. Con is control MEFs, def is Becn1 deficient MEFs, and rev is Becn1 revertant MEFs plus beclin 1. C . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -beclin 1, and -actin blots after VPS34 IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. D . Quantification of normalized 32 P-labelled PI(3)P and 32 P-PI(3)P/VPS34 (from IP) from 3 separate experiments and VPS34/actin (from input lanes) from 9 separate experiments (including ). Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0003, p = 0.0226, and p = 0.0002 top to bottom using a one sample t-test (two-tailed). UVRAG and Atg14-associated PI(3)P production is decreased in Becn1 deficient MEFs. E . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -UVRAG, -beclin 1, and -actin blots after UVRAG IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p<0.0001 using a one sample t-test (two-tailed). F . Autoradiograph of 32 P-PI(3)P and anti-VPS34, -Atg14L, -beclin 1, and -actin blots after Atg14L IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0065 using a one sample t-test (two-tailed).
    Figure Legend Snippet: A . Table showing results from PI(3)P ELISA. Average 450 nm absorbance, p-value of comparison to Becn1 deficient MEFs. PI(3)P concentration is determined by comparison to a standard curve. Quantification of 450 nm absorbance of control, Becn1 deficient, and Becn1 revertant MEFs (6,5,6 replicates); statistics using a one-tailed t-test p = 0.0032, 0.0336. B . Anti-beclin 1, and -actin blots of MEF cell lysates. Con is control MEFs, def is Becn1 deficient MEFs, and rev is Becn1 revertant MEFs plus beclin 1. C . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -beclin 1, and -actin blots after VPS34 IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. D . Quantification of normalized 32 P-labelled PI(3)P and 32 P-PI(3)P/VPS34 (from IP) from 3 separate experiments and VPS34/actin (from input lanes) from 9 separate experiments (including ). Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0003, p = 0.0226, and p = 0.0002 top to bottom using a one sample t-test (two-tailed). UVRAG and Atg14-associated PI(3)P production is decreased in Becn1 deficient MEFs. E . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -UVRAG, -beclin 1, and -actin blots after UVRAG IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p<0.0001 using a one sample t-test (two-tailed). F . Autoradiograph of 32 P-PI(3)P and anti-VPS34, -Atg14L, -beclin 1, and -actin blots after Atg14L IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0065 using a one sample t-test (two-tailed).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, One-tailed Test, Autoradiography, Two Tailed Test

    A . Reduction of Atg12 and WIPI2 puncta formation in Becn1 deficient MEFs. DAPI, endogenous Atg12, WIPI2. White dashed boxes represent zoomed regions. Scalebars = 20 µm. ‘con’ is control MEFs, ‘def’ is beclin 1 deficient MEFs. Quantification of an average of 91 cells per condition from three separate experiments (con: 91, 110, 110; def: 64, 84, 87). Bars represent mean +/− s.e.m. (n = 3); Atg12 p = 0.0039; WIPI2 p = 0.0183 using a one-tailed t-test. B . Rab5-CA mutant is mislocalized in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. MEF cells were transfected with Rab5wt, CA or DN-GFP and visualized. White dashed boxes represent zoomed regions. Scale bars = 20 µm. C . PI(3)P is recruited to expanding endosome sites in control MEFs but no endosome expansion associated with Rab5CA-GFP was found in Becn1 deficient MEFs. MEF cells were transfected with Rab5CA-GFP, fixed, stained with PI(3)P antibody and visualized. Scalebars = 10 µm. Quantification of percentage of cells with no rings or rings (at least 30 transfected cells from at least 3 separate experiments). Control MEFs (7 cells with no rings, 37 cells with rings) vs. Becn1 deficient MEFs (36,5) significantly different (p<0.0001) and Becn1 deficient MEFs (36,6) vs. Becn1 revertant MEFs (11,33) significantly different (p<0.0001) using Fisher's exact test (two-sided).
    Figure Legend Snippet: A . Reduction of Atg12 and WIPI2 puncta formation in Becn1 deficient MEFs. DAPI, endogenous Atg12, WIPI2. White dashed boxes represent zoomed regions. Scalebars = 20 µm. ‘con’ is control MEFs, ‘def’ is beclin 1 deficient MEFs. Quantification of an average of 91 cells per condition from three separate experiments (con: 91, 110, 110; def: 64, 84, 87). Bars represent mean +/− s.e.m. (n = 3); Atg12 p = 0.0039; WIPI2 p = 0.0183 using a one-tailed t-test. B . Rab5-CA mutant is mislocalized in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. MEF cells were transfected with Rab5wt, CA or DN-GFP and visualized. White dashed boxes represent zoomed regions. Scale bars = 20 µm. C . PI(3)P is recruited to expanding endosome sites in control MEFs but no endosome expansion associated with Rab5CA-GFP was found in Becn1 deficient MEFs. MEF cells were transfected with Rab5CA-GFP, fixed, stained with PI(3)P antibody and visualized. Scalebars = 10 µm. Quantification of percentage of cells with no rings or rings (at least 30 transfected cells from at least 3 separate experiments). Control MEFs (7 cells with no rings, 37 cells with rings) vs. Becn1 deficient MEFs (36,5) significantly different (p<0.0001) and Becn1 deficient MEFs (36,6) vs. Becn1 revertant MEFs (11,33) significantly different (p<0.0001) using Fisher's exact test (two-sided).

    Techniques Used: One-tailed Test, Mutagenesis, Transfection, Staining

    anti pi 3 p  (Echelon Biosciences)


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  • 93

    Structured Review

    Echelon Biosciences anti pi 3 p
    A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of <t>PI(3)P</t> and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.
    Anti Pi 3 P, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pi 3 p/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pi 3 p - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex"

    Article Title: Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004626

    A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of PI(3)P and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.
    Figure Legend Snippet: A . L7- Becn1 -eGFP mice were generated using bacterial artificial chromosome (BAC) recombination techniques . Schematic of the Becn1 gene fused to eGFP used to produce beclin 1-GFP in PCs using the L7/Pcp2 gene on BAC. B . beclin 1-GFP and endogenous beclin 1 are expressed in the brain. Anti-beclin 1 blot shows beclin 1-GFP in transgenic (Tg) mice. C . Transgenically expressed beclin 1-GFP is visualized specifically in the PCs of the cerebellum with anti-GFP antibody. Fluorescent image of the cerebellum of an L7- Becn1 -GFP transgenic mouse. D .– F . beclin 1-GFP is found on membrane structures in PCs. Immuno-EM of the cerebellum of P21 L7- Becn1 -GFP transgenic mice showing beclin 1-GFP on endosomes ( D ) and MVBs ( E ),( F ). Scale bars = 250 nm. Loss of beclin 1 in Purkinje cells causes mislocalization of PI(3)P and abnormal endosomes and endolysosomes. G . Endogenous PI(3)P is punctate in Becn1 F/F PCs but diffuse in Becn1 F/F-Pcp2-Cre PCs. Immunofluorescent images of calbindin and PI(3)P stained Becn1 F/F and Becn1 F/F;Pcp2-Cre cerebellums of 1M mice. Dashed white boxes indicate zoomed image. Scale bars = 10 µm. H . GST-FYVE localizes to membranes in Becn1 F/F PCs but is diffuse in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM images of cerebellar slices incubated with GST-FYVE and probed with anti-GST antibody. Scale bars = 200 nm. Arrow indicates endosome. I . EEA1 staining is intense in early endosomal structures in Becn1 F/F control PCs but is reduced significantly on endosomes in Becn1 F/F-Pcp2-Cre PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre PCs stained with EEA1. Scale bars = 500 nm. J . LAMP1-stained vesicles are abnormally shaped and expanded in Becn1 F/F-Pcp2-Cre cKO PCs. Immuno-EM micrographs of P21 Becn1 F/F or Becn1 F/F-Pcp2-Cre cKO PCs stained with LAMP1. Dashed red boxes indicate zoomed image. Scalebars = 500 nm.

    Techniques Used: Generated, Transgenic Assay, Staining, Incubation

    A . Table showing results from PI(3)P ELISA. Average 450 nm absorbance, p-value of comparison to Becn1 deficient MEFs. PI(3)P concentration is determined by comparison to a standard curve. Quantification of 450 nm absorbance of control, Becn1 deficient, and Becn1 revertant MEFs (6,5,6 replicates); statistics using a one-tailed t-test p = 0.0032, 0.0336. B . Anti-beclin 1, and -actin blots of MEF cell lysates. Con is control MEFs, def is Becn1 deficient MEFs, and rev is Becn1 revertant MEFs plus beclin 1. C . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -beclin 1, and -actin blots after VPS34 IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. D . Quantification of normalized 32 P-labelled PI(3)P and 32 P-PI(3)P/VPS34 (from IP) from 3 separate experiments and VPS34/actin (from input lanes) from 9 separate experiments (including ). Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0003, p = 0.0226, and p = 0.0002 top to bottom using a one sample t-test (two-tailed). UVRAG and Atg14-associated PI(3)P production is decreased in Becn1 deficient MEFs. E . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -UVRAG, -beclin 1, and -actin blots after UVRAG IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p<0.0001 using a one sample t-test (two-tailed). F . Autoradiograph of 32 P-PI(3)P and anti-VPS34, -Atg14L, -beclin 1, and -actin blots after Atg14L IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0065 using a one sample t-test (two-tailed).
    Figure Legend Snippet: A . Table showing results from PI(3)P ELISA. Average 450 nm absorbance, p-value of comparison to Becn1 deficient MEFs. PI(3)P concentration is determined by comparison to a standard curve. Quantification of 450 nm absorbance of control, Becn1 deficient, and Becn1 revertant MEFs (6,5,6 replicates); statistics using a one-tailed t-test p = 0.0032, 0.0336. B . Anti-beclin 1, and -actin blots of MEF cell lysates. Con is control MEFs, def is Becn1 deficient MEFs, and rev is Becn1 revertant MEFs plus beclin 1. C . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -beclin 1, and -actin blots after VPS34 IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. D . Quantification of normalized 32 P-labelled PI(3)P and 32 P-PI(3)P/VPS34 (from IP) from 3 separate experiments and VPS34/actin (from input lanes) from 9 separate experiments (including ). Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0003, p = 0.0226, and p = 0.0002 top to bottom using a one sample t-test (two-tailed). UVRAG and Atg14-associated PI(3)P production is decreased in Becn1 deficient MEFs. E . Autoradiograph of 32 P-labelled PI(3)P and anti-VPS34, -UVRAG, -beclin 1, and -actin blots after UVRAG IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Con is control MEFs, def is Becn1 deficient MEFs and def+bec is Becn1 revertant MEFs. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p<0.0001 using a one sample t-test (two-tailed). F . Autoradiograph of 32 P-PI(3)P and anti-VPS34, -Atg14L, -beclin 1, and -actin blots after Atg14L IP of lysates from indicated MEFs with or without wortmannin (Wrtm) treatment. Quantification of normalized 32 P-labelled PI(3)P from 3 separate experiments. Bars represent mean +/− s.e.m. (n = 3) con vs. def p = 0.0065 using a one sample t-test (two-tailed).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, One-tailed Test, Autoradiography, Two Tailed Test

    A . Reduction of Atg12 and WIPI2 puncta formation in Becn1 deficient MEFs. DAPI, endogenous Atg12, WIPI2. White dashed boxes represent zoomed regions. Scalebars = 20 µm. ‘con’ is control MEFs, ‘def’ is beclin 1 deficient MEFs. Quantification of an average of 91 cells per condition from three separate experiments (con: 91, 110, 110; def: 64, 84, 87). Bars represent mean +/− s.e.m. (n = 3); Atg12 p = 0.0039; WIPI2 p = 0.0183 using a one-tailed t-test. B . Rab5-CA mutant is mislocalized in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. MEF cells were transfected with Rab5wt, CA or DN-GFP and visualized. White dashed boxes represent zoomed regions. Scale bars = 20 µm. C . PI(3)P is recruited to expanding endosome sites in control MEFs but no endosome expansion associated with Rab5CA-GFP was found in Becn1 deficient MEFs. MEF cells were transfected with Rab5CA-GFP, fixed, stained with PI(3)P antibody and visualized. Scalebars = 10 µm. Quantification of percentage of cells with no rings or rings (at least 30 transfected cells from at least 3 separate experiments). Control MEFs (7 cells with no rings, 37 cells with rings) vs. Becn1 deficient MEFs (36,5) significantly different (p<0.0001) and Becn1 deficient MEFs (36,6) vs. Becn1 revertant MEFs (11,33) significantly different (p<0.0001) using Fisher's exact test (two-sided).
    Figure Legend Snippet: A . Reduction of Atg12 and WIPI2 puncta formation in Becn1 deficient MEFs. DAPI, endogenous Atg12, WIPI2. White dashed boxes represent zoomed regions. Scalebars = 20 µm. ‘con’ is control MEFs, ‘def’ is beclin 1 deficient MEFs. Quantification of an average of 91 cells per condition from three separate experiments (con: 91, 110, 110; def: 64, 84, 87). Bars represent mean +/− s.e.m. (n = 3); Atg12 p = 0.0039; WIPI2 p = 0.0183 using a one-tailed t-test. B . Rab5-CA mutant is mislocalized in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. MEF cells were transfected with Rab5wt, CA or DN-GFP and visualized. White dashed boxes represent zoomed regions. Scale bars = 20 µm. C . PI(3)P is recruited to expanding endosome sites in control MEFs but no endosome expansion associated with Rab5CA-GFP was found in Becn1 deficient MEFs. MEF cells were transfected with Rab5CA-GFP, fixed, stained with PI(3)P antibody and visualized. Scalebars = 10 µm. Quantification of percentage of cells with no rings or rings (at least 30 transfected cells from at least 3 separate experiments). Control MEFs (7 cells with no rings, 37 cells with rings) vs. Becn1 deficient MEFs (36,5) significantly different (p<0.0001) and Becn1 deficient MEFs (36,6) vs. Becn1 revertant MEFs (11,33) significantly different (p<0.0001) using Fisher's exact test (two-sided).

    Techniques Used: One-tailed Test, Mutagenesis, Transfection, Staining

    ptdins 3 p  (Echelon Biosciences)


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    Echelon Biosciences ptdins 3 p
    Ptdins 3 P, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) <t>PI(3)P</t> levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
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    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) <t>PI(3)P</t> levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
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    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) <t>PI(3)P</t> levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
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    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) <t>PI(3)P</t> levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .
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    TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) PI(3)P levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases

    doi: 10.3389/fcell.2021.709398

    Figure Lengend Snippet: TCR-induced autophagy is dependent upon class I PI3K and 5′ phosphatase activity. (A) AVO formation histograms gated on live CD4 T lymphocytes stimulated with 1ug/mL soluble anti-CD3 and anti-CD28 for 24 and 48 h with the indicated reagents. A 5 mM 3MA, 100 nM PIK75, and 2 μM PIK75 potently reduced AVO formation, especially at 48 h. Histograms are representative of at least 3 independent experiments under each condition. (B) Quantitation of AVO formation from (A) summarizing at least 3 independent experiments per condition. (C) AVO formation in p85 f /f and p85 f /f ER-cre CD4 T cells demonstrates the requirement for class I PI3K in TCR-mediated autophagy induction. Splenocyte and lymphocyte mixtures were cultured for 4 days in 1 ng/mL IL-7 and either 500 nM 4OH Tamoxifen or EtOH, and stimulated for 2 days with 1 μg/mL soluble anti-CD3 and anti-CD28 for 48 h. (D) Quantification of (C) . Ratios of AVO formation of CD4 T cells pre-treated with 4OH Tamoxifen or EtOH to those kept in complete media for 96 h. Data are compiled from 3 independent experiments. (E) p62 degradation and LC3 lipidation are impaired in p85-deficient CD4 T cells. Cells were treated as in (C) . Numbers indicate band densities compared to those of β-actin. (F) PI(3)P levels measured 24 h after activation through soluble anti-CD3 and anti-CD28 as in (C) .

    Article Snippet: Anti-PI(3)P, -PI(3,4)P 2 , -PI(3,4,5)P 3 , and PI(3,4)P 2 lipid were purchased from Echelon Biosciences.

    Techniques: Activity Assay, Quantitation Assay, Cell Culture, Activation Assay

    Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases

    doi: 10.3389/fcell.2021.709398

    Figure Lengend Snippet: Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .

    Article Snippet: Anti-PI(3)P, -PI(3,4)P 2 , -PI(3,4,5)P 3 , and PI(3,4)P 2 lipid were purchased from Echelon Biosciences.

    Techniques: Fluorescence, Microscopy, Quantitation Assay