rat anti mouse lamp2  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Heparan sulfate promotes autoactivation of pro-Cathepsin K by destabilizing the propeptide-catalytic domain interaction

doi: 10.64898/2026.01.18.700217

Figure Lengend Snippet: Mouse femur section was co-stained with anti-CtsK (green), HS20 (purple) and anti-LAMP2 (red) and visualized with anti-rabbit Alexa488, anti-human Alexa594, and anti-rat Alexa647 secondary antibodies, respectively. Area shown is the cortical bone. The approximate boundary of the bone is shown with white dashed line. A blood vessel within the bone is indicated with yellow dashed line. Red blood cells in the vessel shown strong green autofluorescence. A closeup view of the large osteoclast is shown in the right bottom panel. Another osteoclast (likely only the tip of the osteoclast) is indicated with a yellow arrow in the merged image. Nuclei of several osteocytes are indicated with white arrow head. Images representative of three separate experiments.

Article Snippet: Following deparaffinization and citric acid-based antigen retrieval (pH7), sectioned were blocked and incubated overnight at 4 °C with the following primary antibodies: 0.2 μg/ml rabbit anti-mCtsK polyclonal antibody (described previously) , 1ug/ml human anti-HS mAb (HS20, from Bio X cell) , and 1ug/ml Rat anti-mouse Lamp2 (GL2A7, Developmental Studies Hybridoma Bank).

Techniques: Staining

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

Journal: bioRxiv

Article Title: Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

doi: 10.1101/2023.07.12.548710

Figure Lengend Snippet: (A) Confocal microscopy of endogenous BMP (green) and LAMP2 (red) immunofluorescence in WT and R1441G LRRK2 mutant MEF cells. Scale bar: 20µm. (B-C) Quantification of vesicular BMP intensity ( B ) and LAMP2 relative intensity ( C ) per cell area. Colored dots represent mean value from 4 independent experiments and violin plots show the distribution of individual cell data. Significance determined by two-tailed paired t test **p<0.01, ***p<0.001. (D) Representative transmission electron microscopy (TEM) images of Multivesicular endosomes (MVE) from WT and R1441G LRRK2 mutant MEF cells. MVB periphery highlighted in yellow. Scale bar: 250nm. (E) MVE area (µm ) quantification in WT and R1441G LRRK2 mutant cells. Colored dots represent mean values from 3 independent experiments and violin plots show the distribution of individual cell data (35-45 cells/group). (F) Quantification of Intraluminal Vesicles (ILVs) per MVE in WT and R1441G mutant MEF cells. The number of ILVs per MVE are binned in three groups and plotted as a percentage of MVE from the total population of each experiment independently. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test (E) and ordinary two-way ANOVA, uncorrected Fisher’s LSD (F) *p<0.05, ****p<0.0001.

Article Snippet: Primary antibodies diluted in PBS with 1% BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were mouse anti– lysobisphosphatidic acid (anti-BMP) clone 6C4 1:1000 (EMD Millipore; catalog no. MABT837), rat monoclonal anti-mouse LAMP2 1:1000 (Developmental Studies Hybridoma Bank, catalog no.GL2A7), mouse anti-Flotillin1 1:1000 (BD Biosciences; catalog no.610821), rabbit anti-phospho-Rab10 1:1000 (Abcam; catalog no. ab230261), mouse anti-alpha tubulin 1:10000 (SigmaAldrich; catalog no. T5168), mouse anti-Actin 1:5000 (Abcam; catalog no. ab3280).

Techniques: Confocal Microscopy, Immunofluorescence, Mutagenesis, Two Tailed Test, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

doi: 10.1101/2023.07.12.548710

Figure Lengend Snippet: (A) Whole cell lysates from WT and R1441G mutant MEF cells treated with 200nM MLi-2 for 24h were analyzed by western blotting. Representative images of LAMP2, phospho-Rab10 and α- Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. (B) Flow cytometry measurement of GCase activity using PFB-FDGlu fluorescent GCase substrate in WT and R1441G LRRK2 mutant MEF cells treated with 300µM CBE for 24h. (C-D) Whole cell lysates (WCL) and isolated exosomes from WT and R1441G LRRK2 mutant MEF cells treated with 200nM MLi-2 (C) or 300µM CBE (D) for 48h were analyzed by western blotting. Representative images of LAMP2, Flotillin1 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. (E-G) Quantification of LAMP2 and Flotillin1 levels relative to R1441G LRRK2 MEF cells in whole cell lysates (WCL). (E) and isolated exosomes (F-G). Data from 6-9 independent experiments (mean ± SEM). Significance determined by two-tailed paired t test compared to R1441G LRRK2 control *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Primary antibodies diluted in PBS with 1% BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were mouse anti– lysobisphosphatidic acid (anti-BMP) clone 6C4 1:1000 (EMD Millipore; catalog no. MABT837), rat monoclonal anti-mouse LAMP2 1:1000 (Developmental Studies Hybridoma Bank, catalog no.GL2A7), mouse anti-Flotillin1 1:1000 (BD Biosciences; catalog no.610821), rabbit anti-phospho-Rab10 1:1000 (Abcam; catalog no. ab230261), mouse anti-alpha tubulin 1:10000 (SigmaAldrich; catalog no. T5168), mouse anti-Actin 1:5000 (Abcam; catalog no. ab3280).

Techniques: Mutagenesis, Western Blot, Molecular Weight, Marker, Flow Cytometry, Activity Assay, Isolation, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

doi: 10.1101/2023.07.12.548710

Figure Lengend Snippet: (A-C) Quantification of LAMP2 and Flotillin1 levels relative to WT control MEF cells in WCL (A) and isolated exosomes (B-C). Data from 7-8 independent experiments (mean ± SEM). Significance determined by ordinary one-way ANOVA, uncorrected Fisher’s LSD. (D-F) UPLC-MS/MS measurements of cellular (D-E) and exosome-associated (F) BMP isomers normalized to protein content from cells treated with 200nM MLi-2 or 300µM CBE for 48h or left untreated (ctrl). Data shown as fold change relative to WT control MEF cells. Only BMP isomers that were detected are shown. Data from 6-7 independent experiments (mean ± SEM). Significance determined by ordinary two-tailed paired t test.

Article Snippet: Primary antibodies diluted in PBS with 1% BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were mouse anti– lysobisphosphatidic acid (anti-BMP) clone 6C4 1:1000 (EMD Millipore; catalog no. MABT837), rat monoclonal anti-mouse LAMP2 1:1000 (Developmental Studies Hybridoma Bank, catalog no.GL2A7), mouse anti-Flotillin1 1:1000 (BD Biosciences; catalog no.610821), rabbit anti-phospho-Rab10 1:1000 (Abcam; catalog no. ab230261), mouse anti-alpha tubulin 1:10000 (SigmaAldrich; catalog no. T5168), mouse anti-Actin 1:5000 (Abcam; catalog no. ab3280).

Techniques: Control, Isolation, Tandem Mass Spectroscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

doi: 10.1101/2023.07.12.548710

Figure Lengend Snippet: (A) Whole cell lysates (WCL) and isolated exosomes from WT MEF cells treated with 10µM GW4869 or 10nM bafilomycin-A1 (B-A1) for 24h were analyzed by western blotting. Representative images of LAMP2, Flotillin1 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. (B-C) UPLC-MS/MS determination of BMP isomers normalized to protein content from WT MEF cells treated with 10nM B-A1 (B) or 10µM GW4869 (C) for 24h. Data shown as fold change relative to WT control MEF cells. Only BMP isomers that were detected are shown. Data from 6 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D-E) Quantitation of BMP isomers normalized to protein content in exosomes isolated from MEF WT cells treated with 10nM B-A1 (D) or 10µM GW4869 (E) for 24h. Data shown as fold change relative to WT control MEF cells. Only BMP isomers that were detected are shown. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Primary antibodies diluted in PBS with 1% BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were mouse anti– lysobisphosphatidic acid (anti-BMP) clone 6C4 1:1000 (EMD Millipore; catalog no. MABT837), rat monoclonal anti-mouse LAMP2 1:1000 (Developmental Studies Hybridoma Bank, catalog no.GL2A7), mouse anti-Flotillin1 1:1000 (BD Biosciences; catalog no.610821), rabbit anti-phospho-Rab10 1:1000 (Abcam; catalog no. ab230261), mouse anti-alpha tubulin 1:10000 (SigmaAldrich; catalog no. T5168), mouse anti-Actin 1:5000 (Abcam; catalog no. ab3280).

Techniques: Isolation, Western Blot, Molecular Weight, Marker, Tandem Mass Spectroscopy, Control, Two Tailed Test, Quantitation Assay

Journal: bioRxiv

Article Title: Exosome-mediated release of bis(monoacylglycerol)phosphate is regulated by LRRK2 and Glucocerebrosidase activity

doi: 10.1101/2023.07.12.548710

Figure Lengend Snippet: Whole cell lysates (WCL) and isolated exosomes from R1441G LRRK2 MEF cells treated with 10µM GW4869 or 10nM bafilomycin-A1 for 24h were analyzed by western blotting. Representative images of LAMP2, Flotillin1 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa.

Article Snippet: Primary antibodies diluted in PBS with 1% BSA (for immunofluorescence) or 5% skim milk (for immunoblotting) were mouse anti– lysobisphosphatidic acid (anti-BMP) clone 6C4 1:1000 (EMD Millipore; catalog no. MABT837), rat monoclonal anti-mouse LAMP2 1:1000 (Developmental Studies Hybridoma Bank, catalog no.GL2A7), mouse anti-Flotillin1 1:1000 (BD Biosciences; catalog no.610821), rabbit anti-phospho-Rab10 1:1000 (Abcam; catalog no. ab230261), mouse anti-alpha tubulin 1:10000 (SigmaAldrich; catalog no. T5168), mouse anti-Actin 1:5000 (Abcam; catalog no. ab3280).

Techniques: Isolation, Western Blot, Molecular Weight, Marker