antibodies against lamp2  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: GRASP55 Safeguards Proper Lysosome Function by Controlling Sorting of Lysosomal Enzymes at the Golgi

doi: 10.1101/2024.12.10.627846

Figure Lengend Snippet: (A-D) Colocalization analysis of CTSB (A), CTSD (B), PSAP (C), or PGRN (D) with LAMP2 (lysosomal marker) in WT, GRASP55 KO, GRASP65 KO, and GNPTAB KO WI-26 cells, using confocal microscopy. Nuclei stained with DAPI (blue). Magnified insets shown to the right. Scale bars, 10 μm. (E-H) Quantification of colocalization from (A-D). Merged data from 3 independent experiments are shown. n = 70-82 individual cells from 5 independent fields per genotype per experiment. Data in graphs shown as mean ± SD. *** p<0.005, **** p<0.001.

Article Snippet: The H4B4 antibody against LAMP2 was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: GRASP55 Safeguards Proper Lysosome Function by Controlling Sorting of Lysosomal Enzymes at the Golgi

doi: 10.1101/2024.12.10.627846

Figure Lengend Snippet: (A-B) Enlarged lysosome size in GRASP55 KO and GNPTAB KO, but not in GRASP65 KO cells. Immunofluorescence analysis of LIMP-2 and LAMP2 (lysosomal membrane markers) in WT, GRASP55 KO, GRASP65 KO, and GNPTAB KO WI-26 cells, using confocal microscopy. Nuclei stained with DAPI (blue). Magnified insets shown to the right (A). Quantification of relative lysosomal size in (B). Merged data from 4 independent experiments are shown. n = 60-81 individual cells from 5 independent fields per genotype per experiment. (C-D) The enlarged lysosome size is reversed by re-expression of GRASP55 in GRASP55 KO cells. Immunofluorescence analysis of LIMP-2 and LAMP2 (lysosomal membrane markers) in WT, GRASP55 KO, and GRASP55 KO WI-26 cells stably expressing Myc-tagged GRASP55, using confocal microscopy. Nuclei stained with DAPI (blue). Magnified insets shown to the right (C). Quantification of relative lysosomal size in (D). Merged data from 4 independent experiments are shown. n = 50-64 individual cells from 5 independent fields per genotype per experiment. (E) The aberrant lysosomal enzyme secretion is reversed by re-expression of GRASP55 in GRASP55 KO cells. Secretion of lysosomal enzymes (PSAP, CTSD, CTSB) assayed by immunoblotting in the supernatants of WT, GRASP55 KO, and GRASP55 KO WI-26 cells stably expressing Myc-tagged GRASP55. Intracellular levels of PSAP, CTSD, CTSB, GRASP55, and Actin assayed in whole cell lysates. n = 3 independent experiments. Scale bars, 10 μm. Data in graphs shown as mean ± SD. * p<0.05, *** p<0.005. See also Figure S3.

Article Snippet: The H4B4 antibody against LAMP2 was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Immunofluorescence, Membrane, Confocal Microscopy, Staining, Expressing, Stable Transfection, Western Blot

Journal: bioRxiv

Article Title: GRASP55 Safeguards Proper Lysosome Function by Controlling Sorting of Lysosomal Enzymes at the Golgi

doi: 10.1101/2024.12.10.627846

Figure Lengend Snippet: (A-B) Loss of lysosomal mTOR localization in GRASP55 KO and GNPTAB KO cells. Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in WT, GRASP55 KO, GRASP65 KO, and GNPTAB KO WI-26 cells, treated with media containing (+AA) or lacking AAs (–AA) for 2 hours, using confocal microscopy. Nuclei stained with DAPI (blue). Magnified insets shown to the right. Scale bars, 10 μm (A). Quantification of colocalization in (B). Merged data from 3 independent experiments are shown. n = 60 individual cells from 5 independent fields per genotype per experiment. (C-D) Diminished phosphorylation of TFEB/TFE3, but not 4E-BP1, in GRASP55 KO or GNPTAB KO cells. Immunoblots with lysates from WT, GRASP55 KO, GRASP65 KO, and GNPTAB KO WI-26 cells, treated with DMSO (vehicle) or Torin1 (250 nM) for 2 hours, and probed with the indicated antibodies (C). Quantification of TFEB phosphorylation (p-TFEB/Actin ratio) in (D). Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form, S: SUMOylated form. n = 3 independent experiments. (E-F) Nuclear translocation of TFE3 in GRASP55 KO or GNPTAB KO cells. TFE3 localization analysis in WT, GRASP55 KO, GRASP65 KO, and GNPTAB KO WI-26 cells, treated with DMSO (vehicle) or Torin1 (250 nM) for 2 hours, using confocal microscopy. LAMP2 used as lysosomal marker (red). Nuclei stained with DAPI (blue). Magnified insets shown to the right. Scale bars, 20 μm (E). Quantification of % nuclear/cytosolic TFE3 localization in (F). Merged data from 4 independent experiments are shown. n = 80-118 individual cells from 5 independent fields per genotype per experiment. Data in graphs shown as mean ± SD. ** p<0.01, *** p<0.005, **** p<0.001.

Article Snippet: The H4B4 antibody against LAMP2 was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Staining, Western Blot, Translocation Assay

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Lysosomal accumulations of mTOR are lost in concanamycin A (ConA)-treated cells (100 nM, 6 h). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of mTOR/LAMP2 colocalization in (b). n = 50 individual cells from 5 independent fields per condition. (c) ConA treatment (100 nM) preferentially diminishes phosphorylation of the lysosomal substrate TFEB but not of the cytoplasmic substrates S6K and 4E-BP1 under basal culture conditions. ConA (or DMSO as control, Ctrl) was added directly in the media for 6 hours before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (–AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. ConA (or DMSO) was also included in the treatment media. The composition of all media is described in the Methods (see ‘Cell culture treatments’). (d-f) As in (a-c) but for treatments with chloroquine (CQ; 50 μM, 6 h). Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. For all panels, representative data from one out of three independent replicate experiments are shown. Data in graphs shown as mean ± SEM. **** p < 0.0001. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Phospho-proteomics, Control, Lysis, Cell Culture

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal function by BafA1 targeting the v-ATPase. b , c , Basal lysosomal proteolysis in HEK293FT cells shown by accumulation of LC3B upon BafA1 treatment (100 nM, 6 h before fixation) ( b ) and quantification of LC3B signal ( c ). n Ctrl = 49 and n BafA1 = 50 individual cells from five independent fields per condition. d , A schematic representation of the treatment strategy followed in this study, assessing mTORC1 activity under basal (unchallenged cells), starvation or acute re-activation (AA add-back) conditions. AA levels are shown by a black line, and mTORC1 activity by a red line (see also ). e , f , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, treated as indicated, using confocal microscopy (magnified insets shown on the right; scale bars, 25 μm and for insets, 5 μm) ( e ) and quantification of colocalization ( f ). n = 50 individual cells from five independent fields per condition. g , Immunoblots with lysates from HEK293FT WT cells treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and BafA1 as shown, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. For e – g , BafA1 (100 nM) (or DMSO as control, Ctrl) was added directly in the media for 6 h before fixation ( e and f ) or lysis ( g ). For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation or lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. BafA1 (or DMSO) was also included in the treatment media. The composition of all media is described in . Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. and .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Inhibition, Activity Assay, Activation Assay, Marker, Confocal Microscopy, Western Blot, Control, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal proteases by PepA and E64 blocking local AA production. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells ( b ) and its quantification ( c ), treated as indicated, using confocal microscopy. PepA (50 μM) and E64 (25 μM) (or DMSO as control, Ctrl) were added directly in the media for 16 h before fixation (magnified insets shown to the right; scale bars, 25 μm and for insets, 5 μm). n = 56 individual cells from three independent fields per condition. Data shown as mean ± s.e.m. **** P < 0.0001. d , Immunoblots with lysates from HEK293FT WT cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and protease inhibitors (PepA + E64) as shown, probed with the indicated antibodies. PepA (50 μM) and E64 (25 μM) were added directly in the media for 16 h before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. PepA + E64 (or DMSO) were also included in the treatment media. The composition of all media is described in . Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Source numerical data and unprocessed blots are available in .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Inhibition, Blocking Assay, Marker, Confocal Microscopy, Control, Western Blot, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of lysosomal enzyme sorting at the Golgi and delivery to lysosomes that depends on the GNPTAB enzyme. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells using confocal microscopy ( b ) and its quantification ( c ). Cells were transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. The composition of all media is described in . n = 44–50 individual cells from five independent fields per condition (see also ). d , Immunoblots with lysates from HEK293FT WT cells transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions as described in b , probed with the indicated antibodies. e , f , Functional characterization of GNPTAB KO HEK293FT cells. g , h , Lysosomal accumulations of mTOR are lost in GNPTAB KOs ( g ) and quantification of mTOR/LAMP2 colocalization ( h ). n = 50 individual cells from five independent fields per condition. For microscopy, magnified insets are shown to the right. Scale bars, 25 μm and for insets, 5 μm. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Transfection, Control, Western Blot, Functional Assay, Microscopy

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , b , mTOR/LAMP2 colocalization ( a ) and its quantification ( b ). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right ( a ). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also ). c – e , Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB phosphorylation, whereas that of S6K/4E-BP1 remains largely unaffected even at much later timepoints ( c ). Quantification of TFEB phosphorylation in ( d ) and S6K phosphorylation in ( e ). f , g , The rapamycin time course (20 nM, 1–30 min) in control (WT) and RagA/B KO cells, assessing S6K dephosphorylation kinetics ( f ) and the quantification of S6K phosphorylation ( g ). The rate of S6K dephosphorylation is similar between Rag-proficient and Rag-deficient cells. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Blocking Assay, De-Phosphorylation Assay, Phospho-proteomics, Control

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model for the genetic removal of the Rag GTPases. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT and RagA/B KO cells ( b ) and its quantification ( c ), when treated as indicated, using confocal microscopy. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . Magnified insets shown to the right in b . Scale bars, 25 μm and for insets, 5 μm. n = 55–60 individual cells from three or four independent fields per condition (see also ). d , Lyso-IP experiments with WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes were immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in lysosomal and non-lysosomal fractions as well as in whole-cell lysates was analysed by immunoblotting. e , Immuno-EM analysis of mTOR localization. Control (WT) or RagA/B KO MEFs, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, treated as described in b , were stained with antibodies against endogenous mTOR (10 nm gold particles) and LAMP2 (5 nm gold particles) ( e ). Magnified insets shown on the right side; the area used for magnification is marked with a white square. Scale bars, 500 nm and for insets, 80 nm. LY, LAMP2-positive lysosomes. f , g , Quantification of mTOR localization at lysosomes or the cytoplasm in WT ( f ) or RagA/B KO MEFs ( g ), treated and analysed by immuno-EM as in e . Samples incubated with secondary antibodies only (no primary ab) were used as negative controls for background staining. Values represent number of gold particles per μm 2 . n WT = 58–60 ( f ), n KO = 60 ( g ) randomly selected areas (1 μm 2 each) from three independent grids per condition. Data shown as mean ± s.e.m. * P < 0.05, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Fig. .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Stable Transfection, Expressing, Negative Control, Western Blot, Control, Staining, Incubation

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT or RagC/D KO cells, treated as indicated in the figure, using confocal microscopy. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of colocalization in (b). n WT(+AA) = 50, n CDKO(+AA) = 50, n WT(–AA) = 48, n CDKO(–AA) = 49, n WT(–/+AA) = 49, n CDKO(–/+AA) = 50 individual cells from 5 independent fields per condition. Representative data from one out of three independent experiments are shown. (c) Immunoblots with lysates from HEK293FT WT and RagC/D KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. Treatments were performed as in (a). n = 3 independent experiments. (d-e) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in WT or RagA/B KO MEF cells, treated as indicated in the figure, using confocal microscopy. Treatments were performed as in (a). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (d). Quantification of colocalization in (e). n WT(+AA) = 51, n ABKO(+AA) = 50, n WT(–AA) = 49, n ABKO(–AA) = 50, n WT(–/+AA) = 49, n ABKO(–/+AA) = 49 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown. (f) As in (c), but with WT and RagA/B KO MEFs. n = 3 independent experiments. (g) As in (c), but with WT and RagA/B KO SW-620 cells. n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form; S: SUMOylated form. Data in graphs shown as mean ± SEM. *** p < 0.001, **** p < 0.0001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , Immunoblots with lysates from HEK293FT WT and RagA/B KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . b , In vitro kinase assays with mTORC1 immunopurified from WT or RagA/B KO HEK293FT cells and recombinant 4E-BP1 protein used as substrate, with 4E-BP1 phosphorylation detected by immunoblotting. No ATP samples (−ATP) used as negative controls. c , Lyso-IP experiments in WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in the lysosomal and non-lysosomal fractions, as well as in whole-cell lysates, analysed by immunoblotting. Note the absence of S6K from lysosomal fractions and the presence of phospho-TFEB in the lysosomal fractions only of control cells. n = 2 independent experiments. d , e , Phosphorylation of multiple mTORC1 substrates is largely unaffected by BafA1 treatment (100 nM, 6 h) ( d ) or loss of Rag GTPases ( e ). In e , Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. f , g , GRASP55 phosphorylation by mTORC1 is retained in RagA/B KO ( f ) or BafA1-treated cells (100 nM, 6 h) ( g ), similarly to that of S6K. In g , starvation was performed as in a . Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. h , RagC is an additional lysosomal mTORC1 substrate that requires properly functioning lysosomes for its phosphorylation, similarly to TFEB/TFE3. AA starvation or blockage of lysosomal function with BafA1 (100 nM, 6 h) decrease RagC phosphorylation (shown as elevated RagC signal with #5466). Treatments performed as in a . i , j , Lysosomal localization of RagC is unaffected by BafA1 treatment (100 nM, 6 h) ( i ). Quantification of RagC/LAMP2 colocalization in ( j ). Scale bars, 25 μm and for insets, 5 μm. n = 50 individual cells from five independent fields per condition. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. – .

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Western Blot, Lysis, In Vitro, Recombinant, Phospho-proteomics, Stable Transfection, Expressing, Negative Control, Control, Inhibition

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Schematic model of cytoplasmic AA sensing and signaling upstream of the Rags. See text for details. (b-c) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (b). Quantification of colocalization in (c). n siCtrl(+AA) = 49, n siMios(+AA) = 46, n siCtrl(–AA) = 49, n siMios(–AA) = 47, n siCtrl(–/+AA) = 50, n siMios(–/+AA) = 46 individual cells from 5 independent fields per condition. Representative data from one out of two independent experiments are shown. (d) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl), and treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA; 10 or 30 min) conditions, probed with the indicated antibodies. Treatments were performed as in (b). n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. Data in (c) shown as mean ± SEM. * p < 0.05, *** p < 0.001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Marker, Confocal Microscopy, Transfection, Control, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Expression analysis of LAMTOR1 by qPCR confirms successful knockdown in HEK293FT cells. n = 2 independent experiments. (b) Schematic model of lysosomal tethering of the Rag dimer by the LAMTOR complex. (c-d) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl). Magnified insets shown to the right. Scale bars = 10 μm (for insets, 5 μm) (c). Quantification of colocalization in (d). n siCtrl = 50, n siLAMTOR1 = 48 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown as mean ± SEM. **** p < 0.001. (e) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl), cultured under basal conditions, and probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. n = 3 independent experiments. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The H4B4 and ABL-93 antibodies against LAMP2 were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH) and maintained at The University of Iowa, Department of Biology.

Techniques: Expressing, Knockdown, Marker, Confocal Microscopy, Transfection, Control, Western Blot, Cell Culture

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Lysosomal accumulations of mTOR are lost in concanamycin A (ConA)-treated cells (100 nM, 6 h). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of mTOR/LAMP2 colocalization in (b). n = 50 individual cells from 5 independent fields per condition. (c) ConA treatment (100 nM) preferentially diminishes phosphorylation of the lysosomal substrate TFEB but not of the cytoplasmic substrates S6K and 4E-BP1 under basal culture conditions. ConA (or DMSO as control, Ctrl) was added directly in the media for 6 hours before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (–AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. ConA (or DMSO) was also included in the treatment media. The composition of all media is described in the Methods (see ‘Cell culture treatments’). (d-f) As in (a-c) but for treatments with chloroquine (CQ; 50 μM, 6 h). Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. For all panels, representative data from one out of three independent replicate experiments are shown. Data in graphs shown as mean ± SEM. **** p < 0.0001. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Phospho-proteomics, Control, Lysis, Cell Culture

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal function by BafA1 targeting the v-ATPase. b , c , Basal lysosomal proteolysis in HEK293FT cells shown by accumulation of LC3B upon BafA1 treatment (100 nM, 6 h before fixation) ( b ) and quantification of LC3B signal ( c ). n Ctrl = 49 and n BafA1 = 50 individual cells from five independent fields per condition. d , A schematic representation of the treatment strategy followed in this study, assessing mTORC1 activity under basal (unchallenged cells), starvation or acute re-activation (AA add-back) conditions. AA levels are shown by a black line, and mTORC1 activity by a red line (see also ). e , f , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, treated as indicated, using confocal microscopy (magnified insets shown on the right; scale bars, 25 μm and for insets, 5 μm) ( e ) and quantification of colocalization ( f ). n = 50 individual cells from five independent fields per condition. g , Immunoblots with lysates from HEK293FT WT cells treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and BafA1 as shown, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. For e – g , BafA1 (100 nM) (or DMSO as control, Ctrl) was added directly in the media for 6 h before fixation ( e and f ) or lysis ( g ). For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation or lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. BafA1 (or DMSO) was also included in the treatment media. The composition of all media is described in . Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. and .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Activity Assay, Activation Assay, Marker, Confocal Microscopy, Western Blot, Control, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of the pharmacological inhibition of lysosomal proteases by PepA and E64 blocking local AA production. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells ( b ) and its quantification ( c ), treated as indicated, using confocal microscopy. PepA (50 μM) and E64 (25 μM) (or DMSO as control, Ctrl) were added directly in the media for 16 h before fixation (magnified insets shown to the right; scale bars, 25 μm and for insets, 5 μm). n = 56 individual cells from three independent fields per condition. Data shown as mean ± s.e.m. **** P < 0.0001. d , Immunoblots with lysates from HEK293FT WT cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, and protease inhibitors (PepA + E64) as shown, probed with the indicated antibodies. PepA (50 μM) and E64 (25 μM) were added directly in the media for 16 h before lysis. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation or lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. PepA + E64 (or DMSO) were also included in the treatment media. The composition of all media is described in . Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Inhibition, Blocking Assay, Marker, Confocal Microscopy, Control, Western Blot, Lysis

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model of lysosomal enzyme sorting at the Golgi and delivery to lysosomes that depends on the GNPTAB enzyme. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells using confocal microscopy ( b ) and its quantification ( c ). Cells were transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 10 or 30 min. The composition of all media is described in . n = 44–50 individual cells from five independent fields per condition (see also ). d , Immunoblots with lysates from HEK293FT WT cells transiently transfected with siRNAs targeting GNPTAB or a control RNAi duplex (siCtrl) and treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions as described in b , probed with the indicated antibodies. e , f , Functional characterization of GNPTAB KO HEK293FT cells. g , h , Lysosomal accumulations of mTOR are lost in GNPTAB KOs ( g ) and quantification of mTOR/LAMP2 colocalization ( h ). n = 50 individual cells from five independent fields per condition. For microscopy, magnified insets are shown to the right. Scale bars, 25 μm and for insets, 5 μm. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, **** P < 0.0001. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Western Blot, Functional Assay, Microscopy

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , b , mTOR/LAMP2 colocalization ( a ) and its quantification ( b ). mTOR delocalizes away from lysosomes already after 2 h of BafA1 treatment. Time course of BafA1 treatment (100 nM, 2–8 h) to block lysosomal function in HEK293FT cells. Magnified insets shown to the right ( a ). Scale bars, 25 μm and insets, 5 μm. n = 49–50 individual cells from five independent fields per condition (see also ). c – e , Dephosphorylation kinetics of lysosomal (TFEB) and cytoplasmic (S6K and 4E-BP1) substrates of mTORC1 upon BafA1 treatment (100 nM, 1–8 h) in HEK293FT cells showing a rapid drop in TFEB phosphorylation, whereas that of S6K/4E-BP1 remains largely unaffected even at much later timepoints ( c ). Quantification of TFEB phosphorylation in ( d ) and S6K phosphorylation in ( e ). f , g , The rapamycin time course (20 nM, 1–30 min) in control (WT) and RagA/B KO cells, assessing S6K dephosphorylation kinetics ( f ) and the quantification of S6K phosphorylation ( g ). The rate of S6K dephosphorylation is similar between Rag-proficient and Rag-deficient cells. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Blocking Assay, De-Phosphorylation Assay, Phospho-proteomics, Control

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , A schematic model for the genetic removal of the Rag GTPases. b , c , Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT and RagA/B KO cells ( b ) and its quantification ( c ), when treated as indicated, using confocal microscopy. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before fixation. For AA starvation (−AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . Magnified insets shown to the right in b . Scale bars, 25 μm and for insets, 5 μm. n = 55–60 individual cells from three or four independent fields per condition (see also ). d , Lyso-IP experiments with WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes were immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in lysosomal and non-lysosomal fractions as well as in whole-cell lysates was analysed by immunoblotting. e , Immuno-EM analysis of mTOR localization. Control (WT) or RagA/B KO MEFs, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, treated as described in b , were stained with antibodies against endogenous mTOR (10 nm gold particles) and LAMP2 (5 nm gold particles) ( e ). Magnified insets shown on the right side; the area used for magnification is marked with a white square. Scale bars, 500 nm and for insets, 80 nm. LY, LAMP2-positive lysosomes. f , g , Quantification of mTOR localization at lysosomes or the cytoplasm in WT ( f ) or RagA/B KO MEFs ( g ), treated and analysed by immuno-EM as in e . Samples incubated with secondary antibodies only (no primary ab) were used as negative controls for background staining. Values represent number of gold particles per μm 2 . n WT = 58–60 ( f ), n KO = 60 ( g ) randomly selected areas (1 μm 2 each) from three independent grids per condition. Data shown as mean ± s.e.m. * P < 0.05, **** P < 0.0001. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Fig. .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Stable Transfection, Expressing, Negative Control, Western Blot, Control, Staining, Incubation

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a-b) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT or RagC/D KO cells, treated as indicated in the figure, using confocal microscopy. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (a). Quantification of colocalization in (b). n WT(+AA) = 50, n CDKO(+AA) = 50, n WT(–AA) = 48, n CDKO(–AA) = 49, n WT(–/+AA) = 49, n CDKO(–/+AA) = 50 individual cells from 5 independent fields per condition. Representative data from one out of three independent experiments are shown. (c) Immunoblots with lysates from HEK293FT WT and RagC/D KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. Treatments were performed as in (a). n = 3 independent experiments. (d-e) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in WT or RagA/B KO MEF cells, treated as indicated in the figure, using confocal microscopy. Treatments were performed as in (a). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (d). Quantification of colocalization in (e). n WT(+AA) = 51, n ABKO(+AA) = 50, n WT(–AA) = 49, n ABKO(–AA) = 50, n WT(–/+AA) = 49, n ABKO(–/+AA) = 49 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown. (f) As in (c), but with WT and RagA/B KO MEFs. n = 3 independent experiments. (g) As in (c), but with WT and RagA/B KO SW-620 cells. n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form; S: SUMOylated form. Data in graphs shown as mean ± SEM. *** p < 0.001, **** p < 0.0001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: a , Immunoblots with lysates from HEK293FT WT and RagA/B KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in . b , In vitro kinase assays with mTORC1 immunopurified from WT or RagA/B KO HEK293FT cells and recombinant 4E-BP1 protein used as substrate, with 4E-BP1 phosphorylation detected by immunoblotting. No ATP samples (−ATP) used as negative controls. c , Lyso-IP experiments in WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in the lysosomal and non-lysosomal fractions, as well as in whole-cell lysates, analysed by immunoblotting. Note the absence of S6K from lysosomal fractions and the presence of phospho-TFEB in the lysosomal fractions only of control cells. n = 2 independent experiments. d , e , Phosphorylation of multiple mTORC1 substrates is largely unaffected by BafA1 treatment (100 nM, 6 h) ( d ) or loss of Rag GTPases ( e ). In e , Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. f , g , GRASP55 phosphorylation by mTORC1 is retained in RagA/B KO ( f ) or BafA1-treated cells (100 nM, 6 h) ( g ), similarly to that of S6K. In g , starvation was performed as in a . Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. h , RagC is an additional lysosomal mTORC1 substrate that requires properly functioning lysosomes for its phosphorylation, similarly to TFEB/TFE3. AA starvation or blockage of lysosomal function with BafA1 (100 nM, 6 h) decrease RagC phosphorylation (shown as elevated RagC signal with #5466). Treatments performed as in a . i , j , Lysosomal localization of RagC is unaffected by BafA1 treatment (100 nM, 6 h) ( i ). Quantification of RagC/LAMP2 colocalization in ( j ). Scale bars, 25 μm and for insets, 5 μm. n = 50 individual cells from five independent fields per condition. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. n.s., non-significant. Source numerical data and unprocessed blots are available in . See also Extended Data Figs. – .

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Western Blot, Lysis, In Vitro, Recombinant, Phospho-proteomics, Stable Transfection, Expressing, Negative Control, Control, Inhibition

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Schematic model of cytoplasmic AA sensing and signaling upstream of the Rags. See text for details. (b-c) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl) and treated as indicated. For basal (+AA) conditions, culture media were replaced with +AA treatment media 90 min before fixation. For AA starvation (–AA), culture media were replaced by starvation media 1 h before fixation. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in the Methods (see ‘Cell culture treatments’). Magnified insets shown to the right. Scale bars = 25 μm (for insets, 5 μm) (b). Quantification of colocalization in (c). n siCtrl(+AA) = 49, n siMios(+AA) = 46, n siCtrl(–AA) = 49, n siMios(–AA) = 47, n siCtrl(–/+AA) = 50, n siMios(–/+AA) = 46 individual cells from 5 independent fields per condition. Representative data from one out of two independent experiments are shown. (d) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting Mios or a control RNAi duplex (siCtrl), and treated with media containing or lacking AAs, in basal (+AA), starvation (–AA) or add-back (–/+AA; 10 or 30 min) conditions, probed with the indicated antibodies. Treatments were performed as in (b). n = 3 independent experiments. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. Data in (c) shown as mean ± SEM. * p < 0.05, *** p < 0.001, ns: non-significant. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Marker, Confocal Microscopy, Transfection, Control, Cell Culture, Western Blot

Journal: Nature Cell Biology

Article Title: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources

doi: 10.1038/s41556-024-01523-7

Figure Lengend Snippet: (a) Expression analysis of LAMTOR1 by qPCR confirms successful knockdown in HEK293FT cells. n = 2 independent experiments. (b) Schematic model of lysosomal tethering of the Rag dimer by the LAMTOR complex. (c-d) Colocalization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT cells, using confocal microscopy. Cells were transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl). Magnified insets shown to the right. Scale bars = 10 μm (for insets, 5 μm) (c). Quantification of colocalization in (d). n siCtrl = 50, n siLAMTOR1 = 48 individual cells from 3 independent fields per condition. Representative data from one out of two independent experiments are shown as mean ± SEM. **** p < 0.001. (e) Immunoblots with lysates from HEK293FT WT cells, transiently transfected with siRNAs targeting LAMTOR1 or a control RNAi duplex (siCtrl), cultured under basal conditions, and probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P: phosphorylated form. n = 3 independent experiments. Source numerical data and unprocessed blots are available in source data.

Article Snippet: The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80).

Techniques: Expressing, Knockdown, Marker, Confocal Microscopy, Transfection, Control, Western Blot, Cell Culture