mouse polyclonal antiserum to gfp  (TaKaRa)


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    TaKaRa mouse polyclonal antiserum to gfp
    Mouse Polyclonal Antiserum To Gfp, supplied by TaKaRa, 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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    mouse polyclonal antiserum to gfp  (TaKaRa)


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    TaKaRa mouse polyclonal antiserum to gfp
    Mouse Polyclonal Antiserum To Gfp, supplied by TaKaRa, 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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    Average 93 stars, based on 1 article reviews
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    mouse anti gfp  (TaKaRa)


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    TaKaRa mouse anti gfp
    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 <t>knockdown</t> <t>ATG16L1</t> WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to <t>GFP.</t> N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
    Mouse Anti Gfp, supplied by TaKaRa, 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/mouse anti gfp/product/TaKaRa
    Average 94 stars, based on 1 article reviews
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    mouse anti gfp - by Bioz Stars, 2023-11
    94/100 stars

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    1) Product Images from "Leucine regulates autophagy via acetylation of the mTORC1 component raptor"

    Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16886-2

    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 knockdown ATG16L1 WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to GFP. N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
    Figure Legend Snippet: a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 knockdown ATG16L1 WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to GFP. N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.

    Techniques Used: Activation Assay, Two Tailed Test, Immunostaining, Staining, Transfection, Expressing, Degradation Assay, CRISPR, Transgenic Assay

    mouse polyclonal antibody against gfp jl 8  (TaKaRa)


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    TaKaRa mouse polyclonal antibody against gfp jl 8
    Mouse Polyclonal Antibody Against Gfp Jl 8, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 1 article reviews
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    mouse polyclonal antibody against gfp jl 8 - by Bioz Stars, 2023-11
    96/100 stars

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    mouse anti gfp polyclonal antibody  (TaKaRa)


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    TaKaRa mouse anti gfp polyclonal antibody
    Mouse Anti Gfp Polyclonal Antibody, supplied by TaKaRa, 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/mouse anti gfp polyclonal antibody/product/TaKaRa
    Average 93 stars, based on 1 article reviews
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    mouse anti gfp polyclonal antibody  (TaKaRa)


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    TaKaRa mouse anti gfp polyclonal antibody
    CLP is localized to the surface of the parasites. (A) Parasites exogenously expressing CLP with two C-terminal HA tags were stained for immunofluorescence microscopy using an anti-HA antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue). A bright-field image is shown on the right. (B) To further test CLP’s cell surface localization, CLP was exogenously expressed with an N-terminal <t>GFP</t> tag. Indirect immunofluorescence assays were performed using <t>an</t> <t>anti-GFP</t> antibody (green) without cell permeabilization. Merged images of DAPI staining and bright-field images are shown on the right. Indirect immunofluorescence images are representative of 60 parasites viewed in three independent experiments. (C) Predicted topology of CLP generated with the TOPO2 program. The predicted orientation is based on results from panels A and B and immunofluorescent assays performed in the absence or presence of a permeabilizing reagent ( <xref ref-type=Fig. S1 ) as well as from the structural prediction from Fig. 1B . Predicted transmembrane residues are shown in blue. " width="250" height="auto" />
    Mouse Anti Gfp Polyclonal Antibody, supplied by TaKaRa, 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/mouse anti gfp polyclonal antibody/product/TaKaRa
    Average 93 stars, based on 1 article reviews
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    mouse anti gfp polyclonal antibody - by Bioz Stars, 2023-11
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    1) Product Images from "A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis"

    Article Title: A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis

    Journal: mBio

    doi: 10.1128/mBio.00720-19

    CLP is localized to the surface of the parasites. (A) Parasites exogenously expressing CLP with two C-terminal HA tags were stained for immunofluorescence microscopy using an anti-HA antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue). A bright-field image is shown on the right. (B) To further test CLP’s cell surface localization, CLP was exogenously expressed with an N-terminal GFP tag. Indirect immunofluorescence assays were performed using an anti-GFP antibody (green) without cell permeabilization. Merged images of DAPI staining and bright-field images are shown on the right. Indirect immunofluorescence images are representative of 60 parasites viewed in three independent experiments. (C) Predicted topology of CLP generated with the TOPO2 program. The predicted orientation is based on results from panels A and B and immunofluorescent assays performed in the absence or presence of a permeabilizing reagent ( <xref ref-type=Fig. S1 ) as well as from the structural prediction from Fig. 1B . Predicted transmembrane residues are shown in blue. " title="... localization, CLP was exogenously expressed with an N-terminal GFP tag. Indirect immunofluorescence assays were performed using an ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: CLP is localized to the surface of the parasites. (A) Parasites exogenously expressing CLP with two C-terminal HA tags were stained for immunofluorescence microscopy using an anti-HA antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining (blue). A bright-field image is shown on the right. (B) To further test CLP’s cell surface localization, CLP was exogenously expressed with an N-terminal GFP tag. Indirect immunofluorescence assays were performed using an anti-GFP antibody (green) without cell permeabilization. Merged images of DAPI staining and bright-field images are shown on the right. Indirect immunofluorescence images are representative of 60 parasites viewed in three independent experiments. (C) Predicted topology of CLP generated with the TOPO2 program. The predicted orientation is based on results from panels A and B and immunofluorescent assays performed in the absence or presence of a permeabilizing reagent ( Fig. S1 ) as well as from the structural prediction from Fig. 1B . Predicted transmembrane residues are shown in blue.

    Techniques Used: Expressing, Staining, Immunofluorescence, Microscopy, Generated

    Generation of a CLP calcium-binding mutant and measurement of calcium interaction with WT and mutant CLP by isothermal titration calorimetry (ITC). (A) Phyre2 and SuSPect analyses identified the predicted calcium-binding site composed of D443 and D445 as the most sensitive to mutation (see <xref ref-type=Fig. S2 for analysis and comparison of other predicted calcium-binding sites in CLP). The height and color of the bars shown in the color key indicate the predicted functional impact of mutating the aspartate residue to the amino acids shown at the bottom of the histogram. Long red bars in the histogram indicate that introduction of that particular amino acid would lead to the greatest phenotypic change, while short blue bars have the smallest predicted phenotypic effect. (B) A CLP mutant that has D443 and D445 mutated to alanines (CLP-mut) was generated to investigate the functional effects of calcium binding in CLP. Wild-type CLP and CLP-mut were exogenously expressed with an N-terminal GFP tag. As a negative control, parasites were transfected with an empty vector plasmid (EV). Immunoblotting using an anti-GFP antibody confirmed that there were approximately equal amounts of wild-type CLP and CLP-mut overexpression. GAPDH is shown as a loading control. Fold represents the CLP expression levels between CLP and CLP-mut relative to CLP (=1). The thin black line between CLP and CLP-mut indicates the blot was spliced to remove an empty lane between the samples. (C and D) Results of using ITC to measure the binding of calcium to WT rCLP (C) and mutant rCLP (D). Calcium chloride (1 mM) was added to 14 μM WT rCLP or 14 μM mutant rCLP with 15 injections, 2 μl each, at 25°C. (C) The upper panel shows the heat change per second during the injection of calcium chloride to WT rCLP, and the changes decrease as the binding saturates. The lower panel shows the integrated heat changes after subtracting the heat generated from dilution. The binding K d for the WT rCLP was determined to be 0.729 μM. (D) The raw data (upper panel) suggest that there was no detectable calcium binding for mutant rCLP, and the estimated mutant K d was 1.236 mM. " title="... and CLP-mut were exogenously expressed with an N-terminal GFP tag. As a negative control, parasites were transfected ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Generation of a CLP calcium-binding mutant and measurement of calcium interaction with WT and mutant CLP by isothermal titration calorimetry (ITC). (A) Phyre2 and SuSPect analyses identified the predicted calcium-binding site composed of D443 and D445 as the most sensitive to mutation (see Fig. S2 for analysis and comparison of other predicted calcium-binding sites in CLP). The height and color of the bars shown in the color key indicate the predicted functional impact of mutating the aspartate residue to the amino acids shown at the bottom of the histogram. Long red bars in the histogram indicate that introduction of that particular amino acid would lead to the greatest phenotypic change, while short blue bars have the smallest predicted phenotypic effect. (B) A CLP mutant that has D443 and D445 mutated to alanines (CLP-mut) was generated to investigate the functional effects of calcium binding in CLP. Wild-type CLP and CLP-mut were exogenously expressed with an N-terminal GFP tag. As a negative control, parasites were transfected with an empty vector plasmid (EV). Immunoblotting using an anti-GFP antibody confirmed that there were approximately equal amounts of wild-type CLP and CLP-mut overexpression. GAPDH is shown as a loading control. Fold represents the CLP expression levels between CLP and CLP-mut relative to CLP (=1). The thin black line between CLP and CLP-mut indicates the blot was spliced to remove an empty lane between the samples. (C and D) Results of using ITC to measure the binding of calcium to WT rCLP (C) and mutant rCLP (D). Calcium chloride (1 mM) was added to 14 μM WT rCLP or 14 μM mutant rCLP with 15 injections, 2 μl each, at 25°C. (C) The upper panel shows the heat change per second during the injection of calcium chloride to WT rCLP, and the changes decrease as the binding saturates. The lower panel shows the integrated heat changes after subtracting the heat generated from dilution. The binding K d for the WT rCLP was determined to be 0.729 μM. (D) The raw data (upper panel) suggest that there was no detectable calcium binding for mutant rCLP, and the estimated mutant K d was 1.236 mM.

    Techniques Used: Binding Assay, Mutagenesis, Isothermal Titration Calorimetry, Functional Assay, Generated, Negative Control, Transfection, Plasmid Preparation, Western Blot, Over Expression, Expressing, Injection

    mouse anti gfp  (TaKaRa)


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    TaKaRa mouse anti gfp
    Raptor Acetylation <t>by</t> <t>EP300</t> Is Important for mTORC1 Regulation (A) Interaction of EP300 with components of the mTORC1 using <t>GFP-trap</t> or immunoprecipitation (IP) with EP300 antibody. GFP- or YFP-tagged mTORC1 constructs (GFP vector, YFP-mTOR, YFP-Raptor, YFP-proline-rich Akt substrate of 40 kDa [PRAS40], and YFP-DEP domain-containing mTOR-interacting protein [DEPTOR]) were used. Asterisks indicate the predicted position of mTOR, PRAS40, or DEPTOR. HC, heavy immunoglobulin G (IgG) chain; LC, light IgG chain. N = 3. (B) Decreased acetylation of Raptor under AA-starved conditions independently of interaction with mTOR. Acetylated mTOR level was not altered by AA starvation. ∗∗ p < 0.01 versus control cells. N = 3. (C) Acetylation of Raptor by EP300, not by KAT2A or KAT2B. ∗∗∗ p < 0.001 versus control cells. N = 3. (D) Raptor K1097R mutant (KR) is not acetylated. WT, wild-type; KR, K1097R mutant. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus Raptor WT-expressing control cells. N = 3. (E) Cells were depleted of Raptor with small interfering RNA (siRNA) and reconstituted with Raptor WT or KR, then analyzed for mTORC1 activity in the presence or absence of AA, the latter with/without AcCoA in HeLa cells. ∗ p < 0.05, ∗∗ p < 0.01 versus Raptor WT-expressing control cells; # p < 0.05 versus AA-starved cells; & p < 0.05 versus Raptor KR-expressing cells (two-tailed t test); ns, not significant. N = 3. (F and G) Reduced interaction of Raptor KR mutant with the Rag complex using GFP-trap (F) or IP with Flag antibody (G). N = 3. (H) mTORC1 distribution onto lysosomal membranes in HeLa cells depleted of Raptor then reconstituted with Raptor WT or Raptor KR. Scale bars, 10 μm and 2 μm (enlarged images). n = about 40 cells. ∗∗ p < 0.01 versus Raptor WT-expressing control cells. (I and J) Decreased AcCoA levels and acetylation of Raptor in fasted mice brains, livers, and muscles. After 22.5 hr starvation, mice were given free access to food for 1.5 hr followed by a second round of starvation for another 22.5 hr. The tissue samples from fed (n = 5) and fasted mice (n = 6) were analyzed for AcCoA (I) and acetylated Raptor (J) levels. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus fed mice.
    Mouse Anti Gfp, supplied by TaKaRa, 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/mouse anti gfp/product/TaKaRa
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti gfp - by Bioz Stars, 2023-11
    94/100 stars

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    1) Product Images from "Leucine Signals to mTORC1 via Its Metabolite Acetyl-Coenzyme A"

    Article Title: Leucine Signals to mTORC1 via Its Metabolite Acetyl-Coenzyme A

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2018.08.013

    Raptor Acetylation by EP300 Is Important for mTORC1 Regulation (A) Interaction of EP300 with components of the mTORC1 using GFP-trap or immunoprecipitation (IP) with EP300 antibody. GFP- or YFP-tagged mTORC1 constructs (GFP vector, YFP-mTOR, YFP-Raptor, YFP-proline-rich Akt substrate of 40 kDa [PRAS40], and YFP-DEP domain-containing mTOR-interacting protein [DEPTOR]) were used. Asterisks indicate the predicted position of mTOR, PRAS40, or DEPTOR. HC, heavy immunoglobulin G (IgG) chain; LC, light IgG chain. N = 3. (B) Decreased acetylation of Raptor under AA-starved conditions independently of interaction with mTOR. Acetylated mTOR level was not altered by AA starvation. ∗∗ p < 0.01 versus control cells. N = 3. (C) Acetylation of Raptor by EP300, not by KAT2A or KAT2B. ∗∗∗ p < 0.001 versus control cells. N = 3. (D) Raptor K1097R mutant (KR) is not acetylated. WT, wild-type; KR, K1097R mutant. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus Raptor WT-expressing control cells. N = 3. (E) Cells were depleted of Raptor with small interfering RNA (siRNA) and reconstituted with Raptor WT or KR, then analyzed for mTORC1 activity in the presence or absence of AA, the latter with/without AcCoA in HeLa cells. ∗ p < 0.05, ∗∗ p < 0.01 versus Raptor WT-expressing control cells; # p < 0.05 versus AA-starved cells; & p < 0.05 versus Raptor KR-expressing cells (two-tailed t test); ns, not significant. N = 3. (F and G) Reduced interaction of Raptor KR mutant with the Rag complex using GFP-trap (F) or IP with Flag antibody (G). N = 3. (H) mTORC1 distribution onto lysosomal membranes in HeLa cells depleted of Raptor then reconstituted with Raptor WT or Raptor KR. Scale bars, 10 μm and 2 μm (enlarged images). n = about 40 cells. ∗∗ p < 0.01 versus Raptor WT-expressing control cells. (I and J) Decreased AcCoA levels and acetylation of Raptor in fasted mice brains, livers, and muscles. After 22.5 hr starvation, mice were given free access to food for 1.5 hr followed by a second round of starvation for another 22.5 hr. The tissue samples from fed (n = 5) and fasted mice (n = 6) were analyzed for AcCoA (I) and acetylated Raptor (J) levels. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus fed mice.
    Figure Legend Snippet: Raptor Acetylation by EP300 Is Important for mTORC1 Regulation (A) Interaction of EP300 with components of the mTORC1 using GFP-trap or immunoprecipitation (IP) with EP300 antibody. GFP- or YFP-tagged mTORC1 constructs (GFP vector, YFP-mTOR, YFP-Raptor, YFP-proline-rich Akt substrate of 40 kDa [PRAS40], and YFP-DEP domain-containing mTOR-interacting protein [DEPTOR]) were used. Asterisks indicate the predicted position of mTOR, PRAS40, or DEPTOR. HC, heavy immunoglobulin G (IgG) chain; LC, light IgG chain. N = 3. (B) Decreased acetylation of Raptor under AA-starved conditions independently of interaction with mTOR. Acetylated mTOR level was not altered by AA starvation. ∗∗ p < 0.01 versus control cells. N = 3. (C) Acetylation of Raptor by EP300, not by KAT2A or KAT2B. ∗∗∗ p < 0.001 versus control cells. N = 3. (D) Raptor K1097R mutant (KR) is not acetylated. WT, wild-type; KR, K1097R mutant. ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus Raptor WT-expressing control cells. N = 3. (E) Cells were depleted of Raptor with small interfering RNA (siRNA) and reconstituted with Raptor WT or KR, then analyzed for mTORC1 activity in the presence or absence of AA, the latter with/without AcCoA in HeLa cells. ∗ p < 0.05, ∗∗ p < 0.01 versus Raptor WT-expressing control cells; # p < 0.05 versus AA-starved cells; & p < 0.05 versus Raptor KR-expressing cells (two-tailed t test); ns, not significant. N = 3. (F and G) Reduced interaction of Raptor KR mutant with the Rag complex using GFP-trap (F) or IP with Flag antibody (G). N = 3. (H) mTORC1 distribution onto lysosomal membranes in HeLa cells depleted of Raptor then reconstituted with Raptor WT or Raptor KR. Scale bars, 10 μm and 2 μm (enlarged images). n = about 40 cells. ∗∗ p < 0.01 versus Raptor WT-expressing control cells. (I and J) Decreased AcCoA levels and acetylation of Raptor in fasted mice brains, livers, and muscles. After 22.5 hr starvation, mice were given free access to food for 1.5 hr followed by a second round of starvation for another 22.5 hr. The tissue samples from fed (n = 5) and fasted mice (n = 6) were analyzed for AcCoA (I) and acetylated Raptor (J) levels. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus fed mice.

    Techniques Used: Immunoprecipitation, Construct, Plasmid Preparation, Mutagenesis, Expressing, Small Interfering RNA, Activity Assay, Two Tailed Test


    Figure Legend Snippet:

    Techniques Used: Recombinant, shRNA, Software

    mouse jl8 anti gfp  (TaKaRa)


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    TaKaRa mouse jl8 anti gfp
    Mouse Jl8 Anti Gfp, supplied by TaKaRa, 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/mouse jl8 anti gfp/product/TaKaRa
    Average 94 stars, based on 1 article reviews
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    mouse polyclonal antibodies against gfp  (TaKaRa)


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    TaKaRa mouse polyclonal antibodies against gfp
    ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a <t>polyclonal</t> anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.
    Mouse Polyclonal Antibodies Against Gfp, supplied by TaKaRa, 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/mouse polyclonal antibodies against gfp/product/TaKaRa
    Average 93 stars, based on 1 article reviews
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    mouse polyclonal antibodies against gfp - by Bioz Stars, 2023-11
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    Images

    1) Product Images from "A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome"

    Article Title: A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

    Journal: Nature Communications

    doi: 10.1038/ncomms15940

    ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a polyclonal anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.
    Figure Legend Snippet: ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a polyclonal anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.

    Techniques Used: Binding Assay, Mass Spectrometry, Molecular Weight, Purification, Western Blot, Expressing, Fluorescence

    mouse polyclonal anti gfp antibody  (TaKaRa)


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    TaKaRa mouse polyclonal anti gfp antibody
    Mouse Polyclonal Anti Gfp Antibody, supplied by TaKaRa, 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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    mouse polyclonal antibodies against gfp  (TaKaRa)


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    TaKaRa mouse polyclonal antibodies against gfp
    Strains expressing YscQ M218A from the native promoter and therefore lacking YscQ C do not secrete effectors and YscQ M218A requires YscQ C for localization at the injectisome. YscQ C and YscQ M218A colocalize; increased expression levels of mCherry-YscQ C lead to an increase in spot number, but not spot intensity for EGFP-YscQ M218A . (A) Secretion assay showing the secreted proteins in a wild-type (WT) strain, and YscQ M218A , uncomplemented or complemented in trans with YscQ C , EGFP-YscQ C , or mCherry-YscQ C . (B) Fluorescence micrographs showing the distribution of EGFP-YscQ, EGFP-YscQ C , and EGFP-YscQ M218A , uncomplemented or complemented by YscQ C in trans . (C) Cellular distribution of EGFP-YscQ M218A (expressed from its native promoter on the virulence plasmid, second row) and mCherry-YscQ C (expressed in increasing amounts in trans induced by the given concentrations of arabinose, third row). The overlay (bottom row) displays the colocalization of both YscQ versions. Scale bars, 2 μm. (D) Number of detected EGFP-YscQ M218A foci per bacterium in cells expressing increasing amounts of mCherry-YscQ C (as in (C)). n > 170 cells per condition (see for details). Black lines represent the average number of foci per bacterium; circles represent the number of foci per single bacterium (arranged in groups of ten). ***, p < 0.001. (E) Average number of detected EGFP-YscQ M218A foci per bacterium in relation to the amount of mCherry-YscQ C , as quantified on an immunoblot with a <t>polyclonal</t> anti-YscQ antibody . Data points from left to right: no YscQ C (no plasmid), and increasing amounts of mCherry-YscQ C (no arabinose, 0.001%, 0.002%, 0.004%, 0.01%, 0.02% arabinose, as in (C)). (F) Average intensity of foci for the bacteria analyzed in (D)). Error bars represent standard deviations of all foci. Numerical values and raw data for (D–F) can be found in .
    Mouse Polyclonal Antibodies Against Gfp, supplied by TaKaRa, 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 "Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome"

    Article Title: Composition, Formation, and Regulation of the Cytosolic C-ring, a Dynamic Component of the Type III Secretion Injectisome

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002039

    Strains expressing YscQ M218A from the native promoter and therefore lacking YscQ C do not secrete effectors and YscQ M218A requires YscQ C for localization at the injectisome. YscQ C and YscQ M218A colocalize; increased expression levels of mCherry-YscQ C lead to an increase in spot number, but not spot intensity for EGFP-YscQ M218A . (A) Secretion assay showing the secreted proteins in a wild-type (WT) strain, and YscQ M218A , uncomplemented or complemented in trans with YscQ C , EGFP-YscQ C , or mCherry-YscQ C . (B) Fluorescence micrographs showing the distribution of EGFP-YscQ, EGFP-YscQ C , and EGFP-YscQ M218A , uncomplemented or complemented by YscQ C in trans . (C) Cellular distribution of EGFP-YscQ M218A (expressed from its native promoter on the virulence plasmid, second row) and mCherry-YscQ C (expressed in increasing amounts in trans induced by the given concentrations of arabinose, third row). The overlay (bottom row) displays the colocalization of both YscQ versions. Scale bars, 2 μm. (D) Number of detected EGFP-YscQ M218A foci per bacterium in cells expressing increasing amounts of mCherry-YscQ C (as in (C)). n > 170 cells per condition (see for details). Black lines represent the average number of foci per bacterium; circles represent the number of foci per single bacterium (arranged in groups of ten). ***, p < 0.001. (E) Average number of detected EGFP-YscQ M218A foci per bacterium in relation to the amount of mCherry-YscQ C , as quantified on an immunoblot with a polyclonal anti-YscQ antibody . Data points from left to right: no YscQ C (no plasmid), and increasing amounts of mCherry-YscQ C (no arabinose, 0.001%, 0.002%, 0.004%, 0.01%, 0.02% arabinose, as in (C)). (F) Average intensity of foci for the bacteria analyzed in (D)). Error bars represent standard deviations of all foci. Numerical values and raw data for (D–F) can be found in .
    Figure Legend Snippet: Strains expressing YscQ M218A from the native promoter and therefore lacking YscQ C do not secrete effectors and YscQ M218A requires YscQ C for localization at the injectisome. YscQ C and YscQ M218A colocalize; increased expression levels of mCherry-YscQ C lead to an increase in spot number, but not spot intensity for EGFP-YscQ M218A . (A) Secretion assay showing the secreted proteins in a wild-type (WT) strain, and YscQ M218A , uncomplemented or complemented in trans with YscQ C , EGFP-YscQ C , or mCherry-YscQ C . (B) Fluorescence micrographs showing the distribution of EGFP-YscQ, EGFP-YscQ C , and EGFP-YscQ M218A , uncomplemented or complemented by YscQ C in trans . (C) Cellular distribution of EGFP-YscQ M218A (expressed from its native promoter on the virulence plasmid, second row) and mCherry-YscQ C (expressed in increasing amounts in trans induced by the given concentrations of arabinose, third row). The overlay (bottom row) displays the colocalization of both YscQ versions. Scale bars, 2 μm. (D) Number of detected EGFP-YscQ M218A foci per bacterium in cells expressing increasing amounts of mCherry-YscQ C (as in (C)). n > 170 cells per condition (see for details). Black lines represent the average number of foci per bacterium; circles represent the number of foci per single bacterium (arranged in groups of ten). ***, p < 0.001. (E) Average number of detected EGFP-YscQ M218A foci per bacterium in relation to the amount of mCherry-YscQ C , as quantified on an immunoblot with a polyclonal anti-YscQ antibody . Data points from left to right: no YscQ C (no plasmid), and increasing amounts of mCherry-YscQ C (no arabinose, 0.001%, 0.002%, 0.004%, 0.01%, 0.02% arabinose, as in (C)). (F) Average intensity of foci for the bacteria analyzed in (D)). Error bars represent standard deviations of all foci. Numerical values and raw data for (D–F) can be found in .

    Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Western Blot

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    • mouse polyclonal antiserum to gfp  (TaKaRa)
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    TaKaRa mouse polyclonal antiserum to gfp
    Mouse Polyclonal Antiserum To Gfp, supplied by TaKaRa, 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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    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 <t>knockdown</t> <t>ATG16L1</t> WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to <t>GFP.</t> N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
    Mouse Anti Gfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 <t>knockdown</t> <t>ATG16L1</t> WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to <t>GFP.</t> N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
    Mouse Polyclonal Antibody Against Gfp Jl 8, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti gfp polyclonal antibody  (TaKaRa)
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    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 <t>knockdown</t> <t>ATG16L1</t> WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to <t>GFP.</t> N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
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    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 <t>knockdown</t> <t>ATG16L1</t> WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to <t>GFP.</t> N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.
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    mouse polyclonal antibodies against gfp  (TaKaRa)
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    ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a <t>polyclonal</t> anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.
    Mouse Polyclonal Antibodies Against Gfp, supplied by TaKaRa, 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/mouse polyclonal antibodies against gfp/product/TaKaRa
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    mouse polyclonal antibodies against gfp - by Bioz Stars, 2023-11
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    mouse polyclonal anti gfp antibody  (TaKaRa)
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    TaKaRa mouse polyclonal anti gfp antibody
    ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a <t>polyclonal</t> anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.
    Mouse Polyclonal Anti Gfp Antibody, supplied by TaKaRa, 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/mouse polyclonal anti gfp antibody/product/TaKaRa
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    mouse polyclonal anti gfp antibody - by Bioz Stars, 2023-11
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    a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 knockdown ATG16L1 WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to GFP. N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.

    Journal: Nature Communications

    Article Title: Leucine regulates autophagy via acetylation of the mTORC1 component raptor

    doi: 10.1038/s41467-020-16886-2

    Figure Lengend Snippet: a Leu metabolic pathway. Red box shows MCCC1 gene. b Autophagy activation by MCCC1 depletion. siRNA knockdown of MCCC1 in HeLa cells was used to determine whether MCCC1 can regulate autophagy (Con; scrambled, nontargeting siRNA, MCCC1; SMARTpool MCCC1 targeted siRNA). Blots are representative of four independent experiments ( N = 4). Two-tailed unpaired t -test. c Immunostaining of HeLa cells treated with MCCC1 SMARTpool siRNA using MCCC1 (red) and LC3 (green) antibodies, nuclei are stained with DAPI (blue). # MCCC1 knockdown cells, *non-knockdown cells. Scale bar, 10 μm. N = 4; 70–80 cells scored per condition per experiment. Two-tailed unpaired t -test. d Reduced HTT (Q74) aggregation in MCCC1 knockdown HeLa cells. HeLa cells were seeded on coverslips in triplicates, transfected with siRNAs targeting control or MCCC1, followed by HA-tagged HTT (Q74) expression. N = 4, 40–60 cells scored per condition per experiment. Two-tailed unpaired t -test. e α-synuclein degradation assay in control or MCCC1 knockdown ATG16L1 WT or CRISPR KO HeLa cells. Cells were transfected with control or MCCC1 siRNAs for 3 days. In the last 24 h, cells were transfected with empty pEGFP (as a “transfection/loading control”) + pEGFP-α-synuclein A53T. Levels of α-synuclein A53T are expressed as a ratio to GFP. N = 3. * p < 0.05 vs. control cells; two-tailed unpaired t -test. Autophagy activation by MCCC1 depletion in SH-SY5Y cells ( f ) and primary neurons ( g ). N = 3. *** p < 0.001 vs. control cells; two-tailed unpaired t -test. Long exposure (LE); Short exposure (SE). h Autophagic flux in mouse primary cortical neurons from mRFP-GFP-LC3 (tfLC3) transgenic mice. Representative confocal z-stack images (right panel) and total number of GFP/mRFP dots (autophagosomes) and mRFP-only dots (autolysosomes). In total, 25–35 cells analyzed per condition per experiment; two-tailed unpaired t -test. Scale bar, 10 μm. Data are presented as mean values ± SEM. Source data are provided as a file.

    Article Snippet: The following antibodies have been used in this work: mouse anti-Flag M2 (#F3165), rabbit anti-Actin (#A2066), and mouse anti-α-Tubulin (#T9026) from Sigma-Aldrich; mouse anti-GAPDH clone 6C5 (#ab8245), rabbit anti-MCCC1 (#ab178675), rabbit anti-SESN1 (#ab134091), rabbit anti-LC3B (#ab192890), rabbit anti-HMGCL (#ab97293), mouse anti-WIPI2 (#ab105459), rabbit anti-HMGCL (#ab97293), and rabbit anti-AUH (#ab157453) from Abcam; mouse anti-BCKDK (#NBP1-47664), goat anti-HA (#NB600-362), and rabbit anti-MCCC1 (#NBP1-81254) from Novus Biologicals; rabbit anti-EP300 (#sc-585), goat polyclonal antiraptor (#sc-27744), mouse anti-HMGCL (#sc-100548), and mouse anti-MCCC1 (#sc-365754) from SantaCruz Biotechnology; mouse anti-LC3B (#0231-100; Nanotools); rabbit anti-ATG16L1 (#PM040; MBL); mouse anti-GFP (#632375 and #632592; Clontech); mouse anti-HA.11 clone 16B12 (#MMS-101P, Covance); mouse anti-EP300 (#05-257) from Millipore; mouse anti-LAMP1 clone H4A3 (obtained from Developmental Studies Hybridoma Bank, University of Iowa); rabbit anti-LAMP1 (#9091), rabbit anti-mTOR (#2972), rabbit anti-phospho-mTOR (Ser2481; #2972), rabbit antiraptor (#2280), rabbit anti-phospho-p-S6K1 (Thr389; #9234), anti-total S6K1 (#9202), rabbit anti-phospho-S6 Ribosomal Protein (p-S6) (Ser235/236; #4856), rabbit anti-S6 Ribosomal Protein (S6) (#2217), rabbit anti-phospho-4E-BP1 (Thr37/46; #9459), rabbit anti-4E-BP1 (#9452), rabbit anti-phospho-ULK1 (Ser757; #6888), rabbit anti-ULK1 (#4773), rabbit anti-SESN2 (#8487), rabbit anti-LARS (#13868), rabbit anti-PIK3C3 (#4263), rabbit anti-acetylated-Lysine (Ac-K) (#9814, #9441), rabbit anti-RRAGA (#4357), rabbit anti-RRAGB (#8150), rabbit anti-WIPI2 (#8567), and rabbit anti-ATG16L1 (#8089) from Cell Signaling Technology; antimouse (#NA931V) and antirabbit (#NA934V) horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare); antigoat HRP-conjugated secondary antibody (#611620, Invitrogen/Life Technologies).

    Techniques: Activation Assay, Two Tailed Test, Immunostaining, Staining, Transfection, Expressing, Degradation Assay, CRISPR, Transgenic Assay

    ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a polyclonal anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.

    Journal: Nature Communications

    Article Title: A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

    doi: 10.1038/ncomms15940

    Figure Lengend Snippet: ( a ) Schematic depiction of the bound (top) and unbound (bottom) cytosolic complex, colours and full protein names as in e . IM ring is depicted in blue, and export apparatus in green. Presumed connections between the cytosolic complex and the membrane-bound part of the injectisome indicated by dashed lines: (1) YscK or YscQ to IM ring, (2) YscN to export apparatus, possibly via YscO. The absence of YscD prevents binding of the cytosolic complex to the basal body. ( b ) Relative amounts of interaction (as detected in one representative quantitative mass spectrometry experiment, normalized to WT) between the given proteins in strains otherwise WT (black), or strains lacking YscD (blue) or YscN (dark yellow). No int., no interaction detected (YscQ:YscK in strain lacking YscN). ( c ) Interactions of Halo-YscQ (expected molecular weight 69.5 kDa) and YscQ C (expected molecular weight 10.0 kDa), detected in the purification eluate by immunoblot using a polyclonal anti-YscQ antibody. ( d ) Ratio of amounts of interaction (as detected in one representative quantitative mass spectrometry experiment) between non-secreting and secreting conditions for the given proteins (first protein Halo-tagged bait, second protein detected and quantified in eluate by mass spectrometry), corrected for the increase in protein levels under secreting conditions (error bars represent the s.d. in expression levels, as determined by the fluorescence intensity of 60 spots per strain, see for details). ( e ) Schematic representation of the influence of YscD and YscN on the network of stable interactions within the cytosolic T3SS components. There is conflicting evidence on a possible direct interaction of YscN and YscQ, which is not included in this scheme . Dashed line (YscN:YscN interaction), not tested.

    Article Snippet: Secreted proteins were stained using the Coomassie-based ‘Instant blue’ staining solution (Expedeon), while immunoblotting was carried out using mouse polyclonal antibodies against GFP (Clontech 632459; 1:1,000) or rabbit polyclonal antibodies against YscQ (MIPA235; 1:1,000).

    Techniques: Binding Assay, Mass Spectrometry, Molecular Weight, Purification, Western Blot, Expressing, Fluorescence