p01172 Search Results


phospho gcn2 t899  (Boster Bio)
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Boster Bio phospho gcn2 t899
( A , B ) Glutamine removal activates <t>GCN2</t> but not AMPK. Immunoblot from HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in glucose-free DMEM for 2 h, amino acid-free DMEM for 8 h or treated with tunicamycin (TM, 1 μg/ml) for 8 h. n.s. non-specific band. Representative example ( A ) of four biological replicates quantified in ( B ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Q: glutamine, line: average, error bars: standard deviation. ( C , D ) Acute mTORC1 inhibition upon glutamine removal is GATOR1-dependent. Immunoblot from control or DEPDC5 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in leucine-free DMEM for 2 h. Q glutamine, L leucine. n.s.: non-specific bands. Representative example ( C ) of four biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( E , F ) Both acute and late inhibition of mTORC1 to glutamine removal is GCN2-dependent. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( E ) of five biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( G , H ) mTORC1 senses asparagine in a GCN2-dependent manner. Immunoblot from ASNS KO or ASNS GCN2 double KO HEK293T cells incubated in DMEM supplemented with 250 μM asparagine (N) or in regular DMEM, which does not contain asparagine, for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Representative experiment ( G ) of three biological replicates quantified in ( H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Cells supplemented with asparagine (N) for each genotype are set to 1. Line: average, error bars: standard deviation. ( I ) The response of mTORC1 to acute glutamine depletion is mediated by the Rag GTPases, while the response at later time points is Rag-independent. Both Rag-dependent and Rag-independent mechanisms are however downstream of GCN2. .
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( A , B ) Glutamine removal activates GCN2 but not AMPK. Immunoblot from HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in glucose-free DMEM for 2 h, amino acid-free DMEM for 8 h or treated with tunicamycin (TM, 1 μg/ml) for 8 h. n.s. non-specific band. Representative example ( A ) of four biological replicates quantified in ( B ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Q: glutamine, line: average, error bars: standard deviation. ( C , D ) Acute mTORC1 inhibition upon glutamine removal is GATOR1-dependent. Immunoblot from control or DEPDC5 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in leucine-free DMEM for 2 h. Q glutamine, L leucine. n.s.: non-specific bands. Representative example ( C ) of four biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( E , F ) Both acute and late inhibition of mTORC1 to glutamine removal is GCN2-dependent. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( E ) of five biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( G , H ) mTORC1 senses asparagine in a GCN2-dependent manner. Immunoblot from ASNS KO or ASNS GCN2 double KO HEK293T cells incubated in DMEM supplemented with 250 μM asparagine (N) or in regular DMEM, which does not contain asparagine, for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Representative experiment ( G ) of three biological replicates quantified in ( H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Cells supplemented with asparagine (N) for each genotype are set to 1. Line: average, error bars: standard deviation. ( I ) The response of mTORC1 to acute glutamine depletion is mediated by the Rag GTPases, while the response at later time points is Rag-independent. Both Rag-dependent and Rag-independent mechanisms are however downstream of GCN2. .

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A , B ) Glutamine removal activates GCN2 but not AMPK. Immunoblot from HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in glucose-free DMEM for 2 h, amino acid-free DMEM for 8 h or treated with tunicamycin (TM, 1 μg/ml) for 8 h. n.s. non-specific band. Representative example ( A ) of four biological replicates quantified in ( B ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Q: glutamine, line: average, error bars: standard deviation. ( C , D ) Acute mTORC1 inhibition upon glutamine removal is GATOR1-dependent. Immunoblot from control or DEPDC5 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in leucine-free DMEM for 2 h. Q glutamine, L leucine. n.s.: non-specific bands. Representative example ( C ) of four biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( E , F ) Both acute and late inhibition of mTORC1 to glutamine removal is GCN2-dependent. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( E ) of five biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. line: average, error bars: standard deviation. ( G , H ) mTORC1 senses asparagine in a GCN2-dependent manner. Immunoblot from ASNS KO or ASNS GCN2 double KO HEK293T cells incubated in DMEM supplemented with 250 μM asparagine (N) or in regular DMEM, which does not contain asparagine, for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Representative experiment ( G ) of three biological replicates quantified in ( H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Cells supplemented with asparagine (N) for each genotype are set to 1. Line: average, error bars: standard deviation. ( I ) The response of mTORC1 to acute glutamine depletion is mediated by the Rag GTPases, while the response at later time points is Rag-independent. Both Rag-dependent and Rag-independent mechanisms are however downstream of GCN2. .

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Western Blot, Incubation, Control, Protein Concentration, Standard Deviation, Inhibition

( A – H ) mTORC1 inhibition upon acute glutamine depletion for 1 h requires GCN2. Immunoblot from HEK293T ( A , B ), HeLa ( C , D ), U2OS ( E , F ) and HepG2 ( G , H ) cells treated with vehicle (DMSO) or GCN2iB (1 μM) and incubated in regular or glutamine-free DMEM for 1 h. Representative example of 3 ( A ) or 5 ( C , E , G ) biological replicates quantified in ( B , D , F , H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Vehicle-treated unstarved cells are set to 1. Q: glutamine, line: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non significant. ( I , J ) Asparagine supplementation rescues GCN2 activation and mTORC1 inhibition acutely, but not during prolonged glutamine depletion. Immunoblot from control HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points and supplemented with 250 μM asparagine. Representative example ( I ) of 3 biological replicates quantified in ( J ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition is set to 1 and relative phosphorylation of S6K1 upon glutamine starvation for 1 h or glutamine starvation and supplementation of asparagine from one to eight hours is shown. Q: glutamine, N: asparagine, line: average, error bars: standard deviation.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A – H ) mTORC1 inhibition upon acute glutamine depletion for 1 h requires GCN2. Immunoblot from HEK293T ( A , B ), HeLa ( C , D ), U2OS ( E , F ) and HepG2 ( G , H ) cells treated with vehicle (DMSO) or GCN2iB (1 μM) and incubated in regular or glutamine-free DMEM for 1 h. Representative example of 3 ( A ) or 5 ( C , E , G ) biological replicates quantified in ( B , D , F , H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Vehicle-treated unstarved cells are set to 1. Q: glutamine, line: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: non significant. ( I , J ) Asparagine supplementation rescues GCN2 activation and mTORC1 inhibition acutely, but not during prolonged glutamine depletion. Immunoblot from control HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points and supplemented with 250 μM asparagine. Representative example ( I ) of 3 biological replicates quantified in ( J ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition is set to 1 and relative phosphorylation of S6K1 upon glutamine starvation for 1 h or glutamine starvation and supplementation of asparagine from one to eight hours is shown. Q: glutamine, N: asparagine, line: average, error bars: standard deviation.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Inhibition, Western Blot, Incubation, Protein Concentration, Standard Deviation, Activation Assay, Control, Phospho-proteomics

( A ) Graphical scheme of the integrated stress response (ISR) downstream of GCN2. Phosphorylation of eIF2α by GCN2 causes inhibition of eIF2B and, as a consequence, inhibition of canonical translation and increased translation of the transcription factor ATF4. ( B ) qPCR analysis of the transcript levels of selected ATF4 targets from HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine, circle: average, error bars: standard deviation, n = 3 biological replicates. ( C ) Ddit4 upregulation upon glutamine removal is mediated by GCN2. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q glutamine. Representative of three biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( D , E ) Ddit4 contributes to the late, but not the acute response to glutamine removal. Immunoblot from control or Ddit4 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( D ) of six biological replicates quantified in ( E ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( F , G ) Ddit4 mediates Rag-independent mTORC1 inhibition by glutamine deprivation. Immunoblot from control or DEPDC5 Ddit4 double KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( F ) of three biological replicates quantified in ( G ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. .

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A ) Graphical scheme of the integrated stress response (ISR) downstream of GCN2. Phosphorylation of eIF2α by GCN2 causes inhibition of eIF2B and, as a consequence, inhibition of canonical translation and increased translation of the transcription factor ATF4. ( B ) qPCR analysis of the transcript levels of selected ATF4 targets from HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine, circle: average, error bars: standard deviation, n = 3 biological replicates. ( C ) Ddit4 upregulation upon glutamine removal is mediated by GCN2. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q glutamine. Representative of three biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( D , E ) Ddit4 contributes to the late, but not the acute response to glutamine removal. Immunoblot from control or Ddit4 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( D ) of six biological replicates quantified in ( E ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( F , G ) Ddit4 mediates Rag-independent mTORC1 inhibition by glutamine deprivation. Immunoblot from control or DEPDC5 Ddit4 double KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Q glutamine. Representative example ( F ) of three biological replicates quantified in ( G ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. .

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Phospho-proteomics, Inhibition, Incubation, Standard Deviation, Western Blot, Control, Protein Concentration

( A ) Sestrin2 protein levels increase at late time points after glutamine deprivation in a GCN2-dependent manner. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B ) Sestrin2 and Ddit4 protein levels increase upon asparagine deprivation in a GCN2-dependent manner. Immunoblot from ASNS KO or ASNS GCN2 KO HEK293T cells incubated in DMEM supplemented with 250 μM asparagine (N) or in regular DMEM (which does not contain asparagine) for the indicated time points. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( C , D ) Sestrin1/2/3 do not significantly contribute to the inhibition of mTORC1 within the first 8 h of glutamine removal. Immunoblot from control and Sestrin1/2/3 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in leucine-free DMEM for 2 h. Deletion of Sestrin3 was determined through sequencing of the corresponding genomic locus, given the poor quality of the Sestrin3 antibodies tested. Q: glutamine, L: leucine. n.s.: non-specific band. Representative experiment ( B ) of 5 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition is set to 1 for all genotypes. Line: average, error bars: standard deviation. ( E , F ) Sestrin1/2/3 contribute towards maintaining mTORC1 repression upon prolonged removal of glutamine. Immunoblot from control HEK293T and Sestrin1/2/3 KO cells incubated in regular or glutamine-free DMEM overnight (16 h) ( D ) and quantification of Sestrin2 induction at 16 h and at 8 h in the experiment in ( A ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Q: glutamine. Representative of 3 biological replicates. Glutamine-rich condition for each time point is set to 1. Circle: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. ** P < 0.01, *** P < 0.001. ( G ) mTORC1 inhibition upon glutamine depletion requires the TSC complex. Immunoblot from control or TSC2 KO MEF cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with amino acid-free DMEM for 8 h. Q: glutamine. Representative example of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A ) Sestrin2 protein levels increase at late time points after glutamine deprivation in a GCN2-dependent manner. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B ) Sestrin2 and Ddit4 protein levels increase upon asparagine deprivation in a GCN2-dependent manner. Immunoblot from ASNS KO or ASNS GCN2 KO HEK293T cells incubated in DMEM supplemented with 250 μM asparagine (N) or in regular DMEM (which does not contain asparagine) for the indicated time points. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( C , D ) Sestrin1/2/3 do not significantly contribute to the inhibition of mTORC1 within the first 8 h of glutamine removal. Immunoblot from control and Sestrin1/2/3 KO HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were incubated in leucine-free DMEM for 2 h. Deletion of Sestrin3 was determined through sequencing of the corresponding genomic locus, given the poor quality of the Sestrin3 antibodies tested. Q: glutamine, L: leucine. n.s.: non-specific band. Representative experiment ( B ) of 5 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition is set to 1 for all genotypes. Line: average, error bars: standard deviation. ( E , F ) Sestrin1/2/3 contribute towards maintaining mTORC1 repression upon prolonged removal of glutamine. Immunoblot from control HEK293T and Sestrin1/2/3 KO cells incubated in regular or glutamine-free DMEM overnight (16 h) ( D ) and quantification of Sestrin2 induction at 16 h and at 8 h in the experiment in ( A ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Q: glutamine. Representative of 3 biological replicates. Glutamine-rich condition for each time point is set to 1. Circle: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. ** P < 0.01, *** P < 0.001. ( G ) mTORC1 inhibition upon glutamine depletion requires the TSC complex. Immunoblot from control or TSC2 KO MEF cells incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with amino acid-free DMEM for 8 h. Q: glutamine. Representative example of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Western Blot, Control, Incubation, Protein Concentration, Inhibition, Sequencing, Standard Deviation

( A ) Late but not acute mTORC1 inhibition upon glutamine deprivation requires ATF4. Immunoblot from control or ATF4 KO HEK293T cells incubated in regular (without asparagine) or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B , C ) Late but not acute mTORC1 inhibition upon combined glutamine and asparagine deprivation requires ATF4. Immunoblot from control or ATF4 KO HEK293T cells incubated in regular DMEM supplemented with 250 μM asparagine (N) or in glutamine-free DMEM (which does not contain asparagine) for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q: glutamine. Representative example ( B ) of 3 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine- and asparagine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( D ) mTORC1 activity upon glutamine depletion in control or GCN2 KO cells treated with ISRIB. Immunoblot from control or GCN2 KO HEK293T cells treated with vehicle (DMSO) or 200 nM ISRIB and incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine. Representative example of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( E , F ) Acute mTORC1 inhibition upon glutamine removal does not require eIF2α phosphorylation. Immunoblot from control (eIF2α S51S/S ) or knock-in MEF cells harboring a mutation that abolishes eIF2a phosphorylation (eIF2α S51A/A ). Cells were incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with amino acid-free DMEM for 8 h. Q: glutamine. Representative example ( E ) of 3 biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( G , H ) Induction of the ISR by tunicamycin does not inhibit mTORC1 acutely. Immunoblot from control HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points, or treated with vehicle (DMSO) or tunicamycin (TM, 1 μl/ml) for the indicated time points. Q: glutamine. Representative example ( G ) of 3 biological replicates quantified in ( H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. 0 h treatment conditions are set to 1. Line: average, error bars: standard deviation.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A ) Late but not acute mTORC1 inhibition upon glutamine deprivation requires ATF4. Immunoblot from control or ATF4 KO HEK293T cells incubated in regular (without asparagine) or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B , C ) Late but not acute mTORC1 inhibition upon combined glutamine and asparagine deprivation requires ATF4. Immunoblot from control or ATF4 KO HEK293T cells incubated in regular DMEM supplemented with 250 μM asparagine (N) or in glutamine-free DMEM (which does not contain asparagine) for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q: glutamine. Representative example ( B ) of 3 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine- and asparagine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( D ) mTORC1 activity upon glutamine depletion in control or GCN2 KO cells treated with ISRIB. Immunoblot from control or GCN2 KO HEK293T cells treated with vehicle (DMSO) or 200 nM ISRIB and incubated in regular or glutamine-free DMEM for the indicated time points. Q: glutamine. Representative example of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( E , F ) Acute mTORC1 inhibition upon glutamine removal does not require eIF2α phosphorylation. Immunoblot from control (eIF2α S51S/S ) or knock-in MEF cells harboring a mutation that abolishes eIF2a phosphorylation (eIF2α S51A/A ). Cells were incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with amino acid-free DMEM for 8 h. Q: glutamine. Representative example ( E ) of 3 biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( G , H ) Induction of the ISR by tunicamycin does not inhibit mTORC1 acutely. Immunoblot from control HEK293T cells incubated in regular or glutamine-free DMEM for the indicated time points, or treated with vehicle (DMSO) or tunicamycin (TM, 1 μl/ml) for the indicated time points. Q: glutamine. Representative example ( G ) of 3 biological replicates quantified in ( H ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. 0 h treatment conditions are set to 1. Line: average, error bars: standard deviation.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Inhibition, Western Blot, Control, Incubation, Protein Concentration, Standard Deviation, Activity Assay, Phospho-proteomics, Knock-In, Mutagenesis

( A , B ) Acute mTORC1 inhibition after glutamine removal is ATF4-independent. Immunoblot from control or ATF4 KO HEK293T cells stably transfected with ASNS were incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q glutamine. Representative example ( A ) of three biological replicates quantified in ( B ). Glutamine-rich condition for each genotype is set to 1. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Line: average, error bars: standard deviation. ( C , D ) Restoring eIF2B function with ISRIB does not prevent acute mTORC1 inhibition after glutamine removal. Immunoblot from HEK293T cells treated with vehicle (DMSO) or 200 nM ISRIB and incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q glutamine. Representative example ( C ) of four biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for vehicle or ISRIB are set to 1. Line: average, error bars: standard deviation. ( E ) Acute mTORC1 inhibition upon glutamine removal requires GCN2 catalytic activity. Immunoblot from control or GCN2 KO HEK293T cells stably transfected with a HA-tagged negative control protein (HA-metap2), HA-tagged wild-type or kinase-dead (K619R) GCN2 or a GCN2 mutant unable to bind to PP1 (RARA). Cells were incubated in regular or glutamine-free DMEM for 1 h. Q glutamine. Representative of three biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( F , G ) Glutamine removal displaces mTOR from lysosomes in a GCN2-dependent manner. Immunostaining for mTOR and the lysosomal marker LAMP2 in control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for 1 h. Q glutamine. Representative example ( F ) of three biological replicates quantified in ( G ). Data are shown as violin plots of the percentage of LAMP2 signal overlapping with the mTOR signal in each field of view. n = 48 fields of view per condition from three biological replicates (16 fields of view per replicate). Continuous line: median, dashed line: first and third quartiles. Two-way ANOVA and Tukey’s post hoc test. Exact P values are indicated in the figure and highlighted in red when significant (<0.05). Scale bar: 20 µm. ( H ) Graphical scheme of the sequential mechanisms inhibiting mTORC1 in response to glutamine deprivation. At early time points (1–2 h), GCN2 causes inhibition of the Rag GTPases independent of the ISR, likely through phosphorylation of the GATOR complex or of a substrate which in turn impinges on this complex. At intermediate (4–8 h) and late (>8 h) time points, mTORC1 inhibition by GCN2 is instead dependent on induction of the ISR, first through increased expression of Ddit4, which inhibits mTORC1 through the TSC complex in a Rag-independent manner, and then through the leucine sensor Sestrin2, which acts through the GATOR-Rag machinery. .

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A , B ) Acute mTORC1 inhibition after glutamine removal is ATF4-independent. Immunoblot from control or ATF4 KO HEK293T cells stably transfected with ASNS were incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q glutamine. Representative example ( A ) of three biological replicates quantified in ( B ). Glutamine-rich condition for each genotype is set to 1. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Line: average, error bars: standard deviation. ( C , D ) Restoring eIF2B function with ISRIB does not prevent acute mTORC1 inhibition after glutamine removal. Immunoblot from HEK293T cells treated with vehicle (DMSO) or 200 nM ISRIB and incubated in regular or glutamine-free DMEM for the indicated time points. As a control, cells were treated with tunicamycin (TM, 1 μg/ml) for 8 h. Q glutamine. Representative example ( C ) of four biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Glutamine-rich condition for vehicle or ISRIB are set to 1. Line: average, error bars: standard deviation. ( E ) Acute mTORC1 inhibition upon glutamine removal requires GCN2 catalytic activity. Immunoblot from control or GCN2 KO HEK293T cells stably transfected with a HA-tagged negative control protein (HA-metap2), HA-tagged wild-type or kinase-dead (K619R) GCN2 or a GCN2 mutant unable to bind to PP1 (RARA). Cells were incubated in regular or glutamine-free DMEM for 1 h. Q glutamine. Representative of three biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( F , G ) Glutamine removal displaces mTOR from lysosomes in a GCN2-dependent manner. Immunostaining for mTOR and the lysosomal marker LAMP2 in control or GCN2 KO HEK293T cells incubated in regular or glutamine-free DMEM for 1 h. Q glutamine. Representative example ( F ) of three biological replicates quantified in ( G ). Data are shown as violin plots of the percentage of LAMP2 signal overlapping with the mTOR signal in each field of view. n = 48 fields of view per condition from three biological replicates (16 fields of view per replicate). Continuous line: median, dashed line: first and third quartiles. Two-way ANOVA and Tukey’s post hoc test. Exact P values are indicated in the figure and highlighted in red when significant (<0.05). Scale bar: 20 µm. ( H ) Graphical scheme of the sequential mechanisms inhibiting mTORC1 in response to glutamine deprivation. At early time points (1–2 h), GCN2 causes inhibition of the Rag GTPases independent of the ISR, likely through phosphorylation of the GATOR complex or of a substrate which in turn impinges on this complex. At intermediate (4–8 h) and late (>8 h) time points, mTORC1 inhibition by GCN2 is instead dependent on induction of the ISR, first through increased expression of Ddit4, which inhibits mTORC1 through the TSC complex in a Rag-independent manner, and then through the leucine sensor Sestrin2, which acts through the GATOR-Rag machinery. .

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Inhibition, Western Blot, Control, Stable Transfection, Transfection, Incubation, Protein Concentration, Standard Deviation, Activity Assay, Negative Control, Mutagenesis, Immunostaining, Marker, Phospho-proteomics, Expressing

( A ) ARF1 inhibition does not affect mTORC1 activity. Immunoblot from control or GCN2 KO HEK293T cells treated with vehicle (DMSO) or 1 μM Brefeldin A (BFA) for 1 h or starved of glutamine in glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B , C ) Inhibition of mTORC1 in response to acute glutamine removal for 1 h does not require FBXO22. Immunoblot from control or FBXO22 KO HEK293T cells incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative example ( B ) of 3 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. mTOR levels in control unstarved cells are set to 1. Q: glutamine, line: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. n.s.: non significant. ( D ) Inhibition of mTORC1 in response to glutamine removal for 1 h does not require any protein ubiquitination. Immunoblot from control HEK293T cells treated with vehicle (DMSO) or the ubiquitin activating enzyme (UAE) inhibitor TAK-243 (1 μM) and incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A ) ARF1 inhibition does not affect mTORC1 activity. Immunoblot from control or GCN2 KO HEK293T cells treated with vehicle (DMSO) or 1 μM Brefeldin A (BFA) for 1 h or starved of glutamine in glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. ( B , C ) Inhibition of mTORC1 in response to acute glutamine removal for 1 h does not require FBXO22. Immunoblot from control or FBXO22 KO HEK293T cells incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative example ( B ) of 3 biological replicates quantified in ( C ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. mTOR levels in control unstarved cells are set to 1. Q: glutamine, line: average, error bars: standard deviation. One-way ANOVA and Tukey’s post hoc test. n.s.: non significant. ( D ) Inhibition of mTORC1 in response to glutamine removal for 1 h does not require any protein ubiquitination. Immunoblot from control HEK293T cells treated with vehicle (DMSO) or the ubiquitin activating enzyme (UAE) inhibitor TAK-243 (1 μM) and incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Inhibition, Activity Assay, Western Blot, Control, Protein Concentration, Incubation, Standard Deviation, Ubiquitin Proteomics

( A – D ) Phosphoproteomics analysis of control HEK293T and GCN2 KO cells incubated in regular and glutamine-free DMEM for 1 h, as shown in the scheme in ( A ). ( B – D ) Volcano plots comparing the abundance of phosphorylated peptides between the indicated conditions. eIF2β: phospho-eIF2β (S105, T111, S105/T111). mTORC1 substrates: phospho-4EBP1 (S65/T68, S65/T70, T68/T70), phospho-4EBP2 (S65/T70), phospho-S6 (S235/S236). GATOR complex: phospho-DEPDC5 (S503, S1530, S445), phospho-Mios (S766), phospho-Wdr24 (S594/S598), phospho-Wdr59 (S564, S603), phospho-Szt2 (S719, S1642, S1644/1645, S1651, S1656/S1657). Raptor: phospho-Raptor (S722). Dashed lines correspond to p = 0.05 ( x axis) and ±1.2 fold change ( y axis), n = 4 biological replicates. Q: glutamine. ( E ) Inhibition of mTORC1 upon 1 h of glutamine removal does not require phosphorylation of Raptor at Ser721 or Ser722. Immunoblot from control HEK293T cells or Raptor-hypomorphic HEK293T cells that are almost completely lacking Raptor expression and thereby have very low mTORC1 activity. Cells were stably transfected with a HA-tagged control protein (HA-metap2), HA-tagged wild-type Raptor or alanine point mutants abolishing the phosphorylations at S721 and S722, either individually or combined. Cells were incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A – D ) Phosphoproteomics analysis of control HEK293T and GCN2 KO cells incubated in regular and glutamine-free DMEM for 1 h, as shown in the scheme in ( A ). ( B – D ) Volcano plots comparing the abundance of phosphorylated peptides between the indicated conditions. eIF2β: phospho-eIF2β (S105, T111, S105/T111). mTORC1 substrates: phospho-4EBP1 (S65/T68, S65/T70, T68/T70), phospho-4EBP2 (S65/T70), phospho-S6 (S235/S236). GATOR complex: phospho-DEPDC5 (S503, S1530, S445), phospho-Mios (S766), phospho-Wdr24 (S594/S598), phospho-Wdr59 (S564, S603), phospho-Szt2 (S719, S1642, S1644/1645, S1651, S1656/S1657). Raptor: phospho-Raptor (S722). Dashed lines correspond to p = 0.05 ( x axis) and ±1.2 fold change ( y axis), n = 4 biological replicates. Q: glutamine. ( E ) Inhibition of mTORC1 upon 1 h of glutamine removal does not require phosphorylation of Raptor at Ser721 or Ser722. Immunoblot from control HEK293T cells or Raptor-hypomorphic HEK293T cells that are almost completely lacking Raptor expression and thereby have very low mTORC1 activity. Cells were stably transfected with a HA-tagged control protein (HA-metap2), HA-tagged wild-type Raptor or alanine point mutants abolishing the phosphorylations at S721 and S722, either individually or combined. Cells were incubated in regular or glutamine-free DMEM for 1 h. Q: glutamine. Representative of 3 biological replicates. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Phospho-proteomics, Control, Incubation, Inhibition, Western Blot, Expressing, Activity Assay, Stable Transfection, Transfection, Protein Concentration

( A – D ) Removal of most amino acids individually inhibits mTORC1 in a GCN2-dependent manner. Immunoblot from control ( A , B ) or GCN2 KO ( C , D ) HEK293T cells incubated for 1 h in regular DMEM or in DMEM lacking each of the indicated amino acids or all amino acids. Representative examples in ( A , C ) of five independent biological replicates quantified in ( B , D ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Quantifications are expressed as fold change compared to cells incubated in regular DMEM containing all amino acids. Line: average, error bars: standard deviation, n = 5 biological replicates. Exact P values by one-sample t and Wilcoxon test are indicated in the figure and highlighted in red when significant (<0.05). Q glutamine, G glycine, R arginine, C cysteine, H histidine, I isoleucine, L leucine, K lysine, M methionine, F phenylalanine, S serine, T threonine, W tryptophan, Y tyrosine, V valine. ( E – H ) GCN2 and Sestrin1/2/3 contribute both to mTORC1 inhibition upon leucine deprivation. Immunoblot from control, GCN2 KO ( E , F ) or Sestrin1/2/3 triple KO ( G , H ) HEK293T cells incubated in regular DMEM or leucine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Deletion of Sestrin3 was determined through sequencing of the corresponding genomic locus, given the poor quality of the Sestrin3 antibodies tested. L leucine. Representative examples ( E , G ) of multiple biological replicates quantified in ( F , H ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. n = 4 biological replicates for ( E , F ) or three biological replicates for ( G , H ). Leucine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( I , J ) mTORC1 no longer senses leucine removal in GCN2-Sestrin1/2/3 quadruple-knockout cells. Immunoblot from control or GCN2-Sestrin1/2/3 KO HEK293T cells incubated in regular or leucine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. L leucine. Representative example ( I ) of four biological replicates quantified in ( J ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Leucine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. .

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A – D ) Removal of most amino acids individually inhibits mTORC1 in a GCN2-dependent manner. Immunoblot from control ( A , B ) or GCN2 KO ( C , D ) HEK293T cells incubated for 1 h in regular DMEM or in DMEM lacking each of the indicated amino acids or all amino acids. Representative examples in ( A , C ) of five independent biological replicates quantified in ( B , D ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Quantifications are expressed as fold change compared to cells incubated in regular DMEM containing all amino acids. Line: average, error bars: standard deviation, n = 5 biological replicates. Exact P values by one-sample t and Wilcoxon test are indicated in the figure and highlighted in red when significant (<0.05). Q glutamine, G glycine, R arginine, C cysteine, H histidine, I isoleucine, L leucine, K lysine, M methionine, F phenylalanine, S serine, T threonine, W tryptophan, Y tyrosine, V valine. ( E – H ) GCN2 and Sestrin1/2/3 contribute both to mTORC1 inhibition upon leucine deprivation. Immunoblot from control, GCN2 KO ( E , F ) or Sestrin1/2/3 triple KO ( G , H ) HEK293T cells incubated in regular DMEM or leucine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. Deletion of Sestrin3 was determined through sequencing of the corresponding genomic locus, given the poor quality of the Sestrin3 antibodies tested. L leucine. Representative examples ( E , G ) of multiple biological replicates quantified in ( F , H ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. n = 4 biological replicates for ( E , F ) or three biological replicates for ( G , H ). Leucine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( I , J ) mTORC1 no longer senses leucine removal in GCN2-Sestrin1/2/3 quadruple-knockout cells. Immunoblot from control or GCN2-Sestrin1/2/3 KO HEK293T cells incubated in regular or leucine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. L leucine. Representative example ( I ) of four biological replicates quantified in ( J ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Leucine-rich condition for each genotype is set to 1. Line: average, error bars: standard deviation. .

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Western Blot, Control, Incubation, Protein Concentration, Standard Deviation, Inhibition, Sequencing, Quadruple Knockout

( A , B ) Histidine deprivation causes progressive mTORC1 inhibition in an entirely GCN2-dependent manner. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or histidine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. H: arginine. Representative example ( A ) of 3 biological replicates quantified in ( B ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Histidine-replete condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( C , D ) GCN2 is required for complete and persistent mTORC1 inhibition upon arginine deprivation. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or arginine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. R: arginine. Representative example ( C ) of 3 biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Arginine-replete condition for each genotype is set to 1. Line: average, error bars: standard deviation.

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A , B ) Histidine deprivation causes progressive mTORC1 inhibition in an entirely GCN2-dependent manner. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or histidine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. H: arginine. Representative example ( A ) of 3 biological replicates quantified in ( B ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Histidine-replete condition for each genotype is set to 1. Line: average, error bars: standard deviation. ( C , D ) GCN2 is required for complete and persistent mTORC1 inhibition upon arginine deprivation. Immunoblot from control or GCN2 KO HEK293T cells incubated in regular or arginine-free DMEM for the indicated time points. As a control, cells were incubated in amino acid-free DMEM for 8 h. R: arginine. Representative example ( C ) of 3 biological replicates quantified in ( D ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Arginine-replete condition for each genotype is set to 1. Line: average, error bars: standard deviation.

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Inhibition, Western Blot, Control, Incubation, Protein Concentration, Standard Deviation

( A – D ) mTORC1 is reactivated more rapidly upon addback of leucine ( A , B ) than of glutamine ( C , D ). Immunoblot from control HEK293T cells treated either with leucine removal and re-addition ( A , B ) or glutamine removal and re-addition ( C , D ). L leucine, Q glutamine. Representative examples ( A , C ) of three biological replicates quantified in ( B , D ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Unstarved cells are set to 1. Line: average, error bars: standard deviation. ( E , F ) mTORC1 also is reactivated more slowly upon leucine addback in cells lacking Sestrin1/2/3. Sestrin1/2/3 KO HEK293T cells were incubated in regular or leucine-free DMEM for 1 h, or leucine-free DMEM for 1 h and then regular DMEM (with leucine) for the indicated time points. L leucine. Representative example ( E ) of three biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Unstarved cells are set to 1. Line: average, error bars: standard deviation. ( G ) Graphical scheme of the response of mTORC1 to leucine and glutamine re-addition and model of the underlying mechanisms. After addback of leucine, fast release of the inhibition by Sestrin1/2/3 causes partial reactivation of mTORC1, followed by full reactivation only after slower inactivation of GCN2. In contrast, after addback of glutamine, mTORC1 response is entirely driven by inactivation of GCN2 and thus lacks a fast component as in the case of leucine. .

Journal: The EMBO Journal

Article Title: mTORC1 senses glutamine and other amino acids through GCN2

doi: 10.1038/s44318-025-00505-1

Figure Lengend Snippet: ( A – D ) mTORC1 is reactivated more rapidly upon addback of leucine ( A , B ) than of glutamine ( C , D ). Immunoblot from control HEK293T cells treated either with leucine removal and re-addition ( A , B ) or glutamine removal and re-addition ( C , D ). L leucine, Q glutamine. Representative examples ( A , C ) of three biological replicates quantified in ( B , D ), respectively. The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Unstarved cells are set to 1. Line: average, error bars: standard deviation. ( E , F ) mTORC1 also is reactivated more slowly upon leucine addback in cells lacking Sestrin1/2/3. Sestrin1/2/3 KO HEK293T cells were incubated in regular or leucine-free DMEM for 1 h, or leucine-free DMEM for 1 h and then regular DMEM (with leucine) for the indicated time points. L leucine. Representative example ( E ) of three biological replicates quantified in ( F ). The same samples were loaded on different gels and blotted in parallel with the indicated antibodies. Protein concentration was controlled by blotting α-tubulin on a separate gel. Unstarved cells are set to 1. Line: average, error bars: standard deviation. ( G ) Graphical scheme of the response of mTORC1 to leucine and glutamine re-addition and model of the underlying mechanisms. After addback of leucine, fast release of the inhibition by Sestrin1/2/3 causes partial reactivation of mTORC1, followed by full reactivation only after slower inactivation of GCN2. In contrast, after addback of glutamine, mTORC1 response is entirely driven by inactivation of GCN2 and thus lacks a fast component as in the case of leucine. .

Article Snippet: The following primary antibodies were used for immunoblots at 1:1000 dilution: phospho-S6K1 T389 (Cell Signaling Technology #9205), S6K1 (Cell Signaling Technology #2708), phospho-4EBP1 S65 (Cell Signaling Technology #9451), 4EBP1 (Cell Signaling Technology #9452), α-tubulin (Sigma-Aldrich #T9026, 1:5000), ASNS (Cell Signaling Technology #20843), phospho-ACC S79 (Cell Signaling Technology #11818), ACC (Cell Signaling Technology #3662), phospho-Raptor S792 (Cell Signaling Technology #2083), Raptor (Cell Signaling Technology #2280), phospho-GCN2 T899 (Boster # P01172 ), GCN2 (Cell Signaling Technology #3302), phospho-eIF2α S51 (Cell Signaling Technology #3398), eIF2α (Cell Signaling Technology #5324), ATF4 (Cell Signaling Technology #11815), DEPDC5 (Abcam #ab185565), HA tag (Cell Signaling Technology #2367), AMPKα1/α2 (Cell Signaling Technology #2532), Redd1/Ddit4 (Abcam #ab191871), Sestrin1 (Proteintech #21668-1-AP), Sestrin2 (Proteintech #10795-1-AP), FBXO22 (Proteintech #13606-1-AP), mTOR (Cell Signaling Technology #2983), GLUL (Cell Signaling Technology #80636).

Techniques: Western Blot, Control, Protein Concentration, Standard Deviation, Incubation, Inhibition