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vps34 d9a5 atg5  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vps34 d9a5 atg5
    Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for <t>ATG5</t> in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for <t>VPS34</t> (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:
    Vps34 D9a5 Atg5, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vps34 d9a5 atg5/product/Cell Signaling Technology Inc
    Average 94 stars, based on 50 article reviews
    vps34 d9a5 atg5 - by Bioz Stars, 2026-02
    94/100 stars

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    1) Product Images from "Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells"

    Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    Journal: eLife

    doi: 10.7554/elife.14023

    Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:
    Figure Legend Snippet: Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

    Techniques Used: Western Blot, Cell Culture, Staining, Fluorescence, Transduction, shRNA, Control, Transfection

    Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**</=0.001. (D) Flowcytometry based expression of molecules (mean fluorescence intensity, MFI, relative to control isotype antibody shown) enriched in Treg subsets, compared in activated Tregs generated from Cre+ive (open bars) and Cre-ive (black bars) mice. 4–6 mice are included in each group. (E) Flowcytometry plots indicating dilution of CFSE in CD45.2+ (OT-II) gated cells isolated from lymph nodes of mice injected with OT-II cells alone (i) or, OT-II co-injected with Tregs transduced with scrambled (ii) or Notch1 shRNA (iii), three days after antigen challenge. Inset: (ii) confocal images of Tregs detecting Foxp3 in scrambled or Notch1 shRNA groups and (iii) immunoblot for Notch1 in shRNA treated groups. (F) Flowcytometry plots indicating dilution of CFSE in CD45.2+ (OT-II) gated cells isolated from lymph nodes of mice injected with OT-II (i) OT-II + WT Tregs (ii) or OT-II + Notch1-/- Tegs (iii) three days after antigen challenge. (G) CFSE dilutions of OT-II cells co-injected with Notch1-/- Tregs transduced with empty vector (pBABE) (ii) or recombinant NIC (iii) three days after antigen challenge. Data are representative of 2–3 independent experiments with 2–3 mice/ experimental group. Percentage of cells in the CFSE diluted group is indicated in each plot. Tregs activated in vitro are used in all experimetns. (H) Proliferation in CD4+OT-II cells alone (i), or co-injected with Tregs transduced with retroviruses expressing shRNA to VPS34 (iii) or a scrambled control (ii) post antigen challenge. (I) Confocal (merged) images of Foxp3 (green) immunostaining counterstained with Hoechst 33342 (blue). (J) immunoblot detecting VPS34 (H) in shRNA treated groups as in H. (K) Flow cytometry plots indicating CFSE dilution in naı¨ve CD4+T- cells 72 hr post-stimulation with anti-CD3 and APC in vitro. T-cells were either cultured alone (no Tregs) or with Tregs transduced with shRNA as described in H. (L) Percent CFSE positive, CD45.2+ OT-II cells isolated from host mice isolated after antigen challenge. Host mice were injected with OT-II naı¨ve T-cells alone (no Tregs) or, naı¨ve cells co-injected with Notch1-/- Tregs retrovirally transduced with empty vector pBABE or recombinant ATG3. Scale bar: 5 mm. This figure is accompanied by Figure 5—figure supplement 1. DOI: 10.7554/eLife.14023.011 The following figure supplement is available for figure 5:
    Figure Legend Snippet: Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**

    Techniques Used: Activity Assay, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Expressing, Fluorescence, Generated, Isolation, Injection, Transduction, shRNA, Western Blot, Plasmid Preparation, Recombinant, In Vitro, Immunostaining, Flow Cytometry, Cell Culture



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    vps34 d9a5 atg5  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc vps34 d9a5 atg5
    Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for <t>ATG5</t> in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for <t>VPS34</t> (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:
    Vps34 D9a5 Atg5, 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/vps34 d9a5 atg5/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    vps34 d9a5 atg5 - by Bioz Stars, 2026-02
    94/100 stars
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    Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

    Journal: eLife

    Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    doi: 10.7554/elife.14023

    Figure Lengend Snippet: Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

    Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

    Techniques: Western Blot, Cell Culture, Staining, Fluorescence, Transduction, shRNA, Control, Transfection

    Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**</=0.001. (D) Flowcytometry based expression of molecules (mean fluorescence intensity, MFI, relative to control isotype antibody shown) enriched in Treg subsets, compared in activated Tregs generated from Cre+ive (open bars) and Cre-ive (black bars) mice. 4–6 mice are included in each group. (E) Flowcytometry plots indicating dilution of CFSE in CD45.2+ (OT-II) gated cells isolated from lymph nodes of mice injected with OT-II cells alone (i) or, OT-II co-injected with Tregs transduced with scrambled (ii) or Notch1 shRNA (iii), three days after antigen challenge. Inset: (ii) confocal images of Tregs detecting Foxp3 in scrambled or Notch1 shRNA groups and (iii) immunoblot for Notch1 in shRNA treated groups. (F) Flowcytometry plots indicating dilution of CFSE in CD45.2+ (OT-II) gated cells isolated from lymph nodes of mice injected with OT-II (i) OT-II + WT Tregs (ii) or OT-II + Notch1-/- Tegs (iii) three days after antigen challenge. (G) CFSE dilutions of OT-II cells co-injected with Notch1-/- Tregs transduced with empty vector (pBABE) (ii) or recombinant NIC (iii) three days after antigen challenge. Data are representative of 2–3 independent experiments with 2–3 mice/ experimental group. Percentage of cells in the CFSE diluted group is indicated in each plot. Tregs activated in vitro are used in all experimetns. (H) Proliferation in CD4+OT-II cells alone (i), or co-injected with Tregs transduced with retroviruses expressing shRNA to VPS34 (iii) or a scrambled control (ii) post antigen challenge. (I) Confocal (merged) images of Foxp3 (green) immunostaining counterstained with Hoechst 33342 (blue). (J) immunoblot detecting VPS34 (H) in shRNA treated groups as in H. (K) Flow cytometry plots indicating CFSE dilution in naı¨ve CD4+T- cells 72 hr post-stimulation with anti-CD3 and APC in vitro. T-cells were either cultured alone (no Tregs) or with Tregs transduced with shRNA as described in H. (L) Percent CFSE positive, CD45.2+ OT-II cells isolated from host mice isolated after antigen challenge. Host mice were injected with OT-II naı¨ve T-cells alone (no Tregs) or, naı¨ve cells co-injected with Notch1-/- Tregs retrovirally transduced with empty vector pBABE or recombinant ATG3. Scale bar: 5 mm. This figure is accompanied by Figure 5—figure supplement 1. DOI: 10.7554/eLife.14023.011 The following figure supplement is available for figure 5:

    Journal: eLife

    Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    doi: 10.7554/elife.14023

    Figure Lengend Snippet: Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**

    Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

    Techniques: Activity Assay, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Expressing, Fluorescence, Generated, Isolation, Injection, Transduction, shRNA, Western Blot, Plasmid Preparation, Recombinant, In Vitro, Immunostaining, Flow Cytometry, Cell Culture