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rat anti-lamp1 (cd107a) antibody  (Merck & Co)

 
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    Merck & Co rat anti-lamp1 (cd107a) antibody
    Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: <t>anti-LAMP1</t> (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .
    Rat Anti Lamp1 (Cd107a) Antibody, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration"

    Article Title: The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201310043

    Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .
    Figure Legend Snippet: Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .

    Techniques Used: Expressing, Transfection, Migration, Wound Healing Assay

    Absence of p14–MP1 or blockage of Arl8b-dependent late endosomal transport causes IQGAP1 accumulation in FAs. (A) IF: anti-Paxillin and anti-IQGAP1 antibodies. IQGAP1 localizes to the leading edge in p14 f/− (white arrows) and colocalizes with Paxillin in FAs in p14 −/− MEFs (red arrows). (B) IF: anti-Paxillin and anti-IQGAP1 antibodies. Shown are different time points during spreading of p14 f/− and p14 −/− MEFs. White arrows indicate accumulation of IQGAP1 at the leading edge in control MEFs. Red arrows point at IQGAP1 localization at Paxillin-positive FAs in control and p14 −/− MEFs. (C) IF: anti-LAMP1 (green), anti-IQGAP1 (red) antibodies, and Hoechst (blue). IQGAP1 localizes to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) and localizes to FAs upon Arl8b depletion (red arrows). (D) p14 f/− MEFs treated as in C. IF: anti-Paxillin and anti-IQGAP1 antibodies. Note IQGAP1 localization to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) versus colocalization of IQGAP1 and Paxillin in FAs upon ARl8b depletion (red arrows).
    Figure Legend Snippet: Absence of p14–MP1 or blockage of Arl8b-dependent late endosomal transport causes IQGAP1 accumulation in FAs. (A) IF: anti-Paxillin and anti-IQGAP1 antibodies. IQGAP1 localizes to the leading edge in p14 f/− (white arrows) and colocalizes with Paxillin in FAs in p14 −/− MEFs (red arrows). (B) IF: anti-Paxillin and anti-IQGAP1 antibodies. Shown are different time points during spreading of p14 f/− and p14 −/− MEFs. White arrows indicate accumulation of IQGAP1 at the leading edge in control MEFs. Red arrows point at IQGAP1 localization at Paxillin-positive FAs in control and p14 −/− MEFs. (C) IF: anti-LAMP1 (green), anti-IQGAP1 (red) antibodies, and Hoechst (blue). IQGAP1 localizes to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) and localizes to FAs upon Arl8b depletion (red arrows). (D) p14 f/− MEFs treated as in C. IF: anti-Paxillin and anti-IQGAP1 antibodies. Note IQGAP1 localization to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) versus colocalization of IQGAP1 and Paxillin in FAs upon ARl8b depletion (red arrows).

    Techniques Used:



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    Image Search Results


    a Representative confocal images demonstrating abnormally enlarged vesicles in Chmp5 Ctsk relative to wild-type skeletal progenitors. Abnormal cells identified by containing enlarged GFP + vesicles. n = 200 cells from three mice per group. b Schematic showing molecular markers utilized for analyzing the endocytic pathway. c Representative confocal images showing LAMP1 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 20 cells each group. d Quantification of LAMP1 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 93, 66 cells respectively. e Representative confocal images showing RAB7 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 15 cells per genotype. f Quantification of RAB7 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 94, 92 cells respectively. g Co-localization of LAMP1 and RAB7 in ATDC5 cells with or without Chmp5 depletion. n = 20 cells per group. h Transmission electron microscopy (TEM) showing accumulation of multivesicular body (MVB)-like (arrows) and lysosome-like (arrowheads) structures in Chmp5 Ctsk relative to wild-type skeletal progenitors. n = 30 cells per group. i Confocal live-cell images demonstrating delayed degradation of EGF-conjugate in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 10 cells each group per time-point. Data shown as mean ± s.d.; two-tailed Student’s t -test. Scale bars, 10 µm except in ( h ) as indicated.

    Journal: bioRxiv

    Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

    doi: 10.1101/2020.08.03.233874

    Figure Lengend Snippet: a Representative confocal images demonstrating abnormally enlarged vesicles in Chmp5 Ctsk relative to wild-type skeletal progenitors. Abnormal cells identified by containing enlarged GFP + vesicles. n = 200 cells from three mice per group. b Schematic showing molecular markers utilized for analyzing the endocytic pathway. c Representative confocal images showing LAMP1 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 20 cells each group. d Quantification of LAMP1 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 93, 66 cells respectively. e Representative confocal images showing RAB7 immunostaining in Chmp5 Ctsk and wild-type skeletal progenitors. n = 15 cells per genotype. f Quantification of RAB7 + vesicles in ATDC5 cells with or without Chmp5 depletion. n = 94, 92 cells respectively. g Co-localization of LAMP1 and RAB7 in ATDC5 cells with or without Chmp5 depletion. n = 20 cells per group. h Transmission electron microscopy (TEM) showing accumulation of multivesicular body (MVB)-like (arrows) and lysosome-like (arrowheads) structures in Chmp5 Ctsk relative to wild-type skeletal progenitors. n = 30 cells per group. i Confocal live-cell images demonstrating delayed degradation of EGF-conjugate in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 10 cells each group per time-point. Data shown as mean ± s.d.; two-tailed Student’s t -test. Scale bars, 10 µm except in ( h ) as indicated.

    Article Snippet: Rabbit anti-CHMP5 polyclonal antibody Mouse anti-EEA1 antibody (clone 14/EEA1, BD Bioscience) Rabbit anti-RAB7 antibody (clone D95F2, Cell Signaling Technology) Rabbit anti-RAB11 antibody (clone D4F5, Cell Signaling Technology) Rat anti-CD107a (LAMP1) antibody (clone 1D4B, BD Bioscience) Rat anti-TGN38 antibody (clone 831629, R&D Systems) Mouse anti-OXPHOS antibody cocktail (cat#ab110413, Abcam) Rabbit anti-Ki67 antibody (cat# ab15580, Abcam) Mouse anti-GAPDH antibody (clone GT239, GeneTex) Mouse anti-beta-actin antibody (clone 2F1-1, Biolegend) APC anti-mouse CD45 (clone 30-F11, Biolegend) APC anti-mouse CD31 (clone 390, Biolegend) Goat anti-Rat secondary antibody, Alexa Fluor 633 (cat# A-21094, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 633 (cat# A-21050, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 488 (cat# A-11006, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 488 (cat# A-11001, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 594 (cat# A-11007, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 594 (cat# A-11005, ThermoFisher) Goat anti-Rabbit secondary antibody, Alexa Fluor® 594 (cat# A-11012, ThermoFisher) In Situ Apoptosis Detection Kit (cat#ab206386, Abcam) Lysotracker™ Red DND-99 (cat# L7528, ThermoFisher) pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate (cat# P35374, ThermoFisher) Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE, cat# T669, ThermoFisher) MitoTracker™ Green FM (cat# M7514, ThermoFisher) CellROX™ Deep Red Reagent (cat# C10422, ThermoFisher) N-Acetyl-L-cysteine (cat# A7250, Sigma-Aldrich) Quercetin (cat# Q4951, Sigma-Aldrich) Dasatinib (cat# CDS023389, Sigma-Aldrich) Polyethylene glycol 400 (PEG400, Fisher Scientific) Annexin V Apoptosis Detection Kit APC (cat# 88-8007-72, ThermoFisher) APC BrdU Flow Kit (cat# 552598, BD Bioscience) InSolution™ Q-VD-OPh, Non-O-methylated (cat# 551476, Millipore) VECTASTAIN® Elite® ABC HRP Kit (Peroxidase, Rabbit IgG; cat# PK-6101, VECTOR Laboratories) Recombinant Mouse TRANCE/RANK L/TNFSF11 Protein (cat# 462-TR, R&D Systems) Recombinant Mouse M-CSF Protein (cat# 461-ML, R&D Systems) alamarBlue™ Cell Viability Reagent (cat# DAL1025, ThermoFisher) Seahorse XF Cell Mito Stress Test Kit (cat# 103015-100, Agilent) ALIZARIN red 2% solution pH 4.2 (cat# 50-317-34, Fisher scientific) Zolendronic acid monohydrate (cat# SML0223, Sigma-Aldrich) OPG-Fc (Amgen Inc.) MC3T3-E1 cells (mouse C57BL/6 calvarial fibroblasts; cat# 99072810, Sigma-Aldrich) ATDC5 cells (mouse 129 teratocarcinoma-derived osteochondral progenitors; cat# 99072806, Sigma-Aldrich)

    Techniques: Immunostaining, Transmission Assay, Electron Microscopy, Two Tailed Test

    a Representative confocal fluorescence images and quantification of fluorescence intensity of LysoTracker Red DND-99 in Chmp5 Ctsk compared to wild-type skeletal progenitors. n = 11 replicates per group for quantitative analysis; repeated 3 times using cells from 3 mice. Scale bars, 50 µm. b Isotype controls for immunofluorescence staining. Scale bars, 20 µm. c, d Confocal images of LAMP1( c ) and RAB7 ( d ) immunostaining in ATDC5 cells with or without Chmp5 deletion. Scale bars, 20 µm ( c ), 10 µm ( d ). e Additional TEM images showing accumulation of MVB-like (arrows) and lysosome-like (arrowheads) structures in Chmp5- deficient skeletal progenitors. f-h Representative confocal images of immunostaining for early endosome marker EEA1 ( f ), cycling endosome marker RAB11 ( g ), and trans-Golgi network marker TGN38 ( h ) in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 30 cells per group. Scale bars, 10 µm ( e ), 20 µm ( f ), 25 µm ( g ). Data shown as mean ± s.d.; two-tailed Student’s t -test.

    Journal: bioRxiv

    Article Title: The ESCRT protein CHMP5 restrains skeletal progenitor cell senescence by preserving endo-lysosomal-mitochondrial network

    doi: 10.1101/2020.08.03.233874

    Figure Lengend Snippet: a Representative confocal fluorescence images and quantification of fluorescence intensity of LysoTracker Red DND-99 in Chmp5 Ctsk compared to wild-type skeletal progenitors. n = 11 replicates per group for quantitative analysis; repeated 3 times using cells from 3 mice. Scale bars, 50 µm. b Isotype controls for immunofluorescence staining. Scale bars, 20 µm. c, d Confocal images of LAMP1( c ) and RAB7 ( d ) immunostaining in ATDC5 cells with or without Chmp5 deletion. Scale bars, 20 µm ( c ), 10 µm ( d ). e Additional TEM images showing accumulation of MVB-like (arrows) and lysosome-like (arrowheads) structures in Chmp5- deficient skeletal progenitors. f-h Representative confocal images of immunostaining for early endosome marker EEA1 ( f ), cycling endosome marker RAB11 ( g ), and trans-Golgi network marker TGN38 ( h ) in Chmp5 Ctsk versus wild-type skeletal progenitors. n = 30 cells per group. Scale bars, 10 µm ( e ), 20 µm ( f ), 25 µm ( g ). Data shown as mean ± s.d.; two-tailed Student’s t -test.

    Article Snippet: Rabbit anti-CHMP5 polyclonal antibody Mouse anti-EEA1 antibody (clone 14/EEA1, BD Bioscience) Rabbit anti-RAB7 antibody (clone D95F2, Cell Signaling Technology) Rabbit anti-RAB11 antibody (clone D4F5, Cell Signaling Technology) Rat anti-CD107a (LAMP1) antibody (clone 1D4B, BD Bioscience) Rat anti-TGN38 antibody (clone 831629, R&D Systems) Mouse anti-OXPHOS antibody cocktail (cat#ab110413, Abcam) Rabbit anti-Ki67 antibody (cat# ab15580, Abcam) Mouse anti-GAPDH antibody (clone GT239, GeneTex) Mouse anti-beta-actin antibody (clone 2F1-1, Biolegend) APC anti-mouse CD45 (clone 30-F11, Biolegend) APC anti-mouse CD31 (clone 390, Biolegend) Goat anti-Rat secondary antibody, Alexa Fluor 633 (cat# A-21094, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 633 (cat# A-21050, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 488 (cat# A-11006, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 488 (cat# A-11001, ThermoFisher) Goat anti-Rat secondary antibody, Alexa Fluor 594 (cat# A-11007, ThermoFisher) Goat anti-Mouse secondary antibody, Alexa Fluor 594 (cat# A-11005, ThermoFisher) Goat anti-Rabbit secondary antibody, Alexa Fluor® 594 (cat# A-11012, ThermoFisher) In Situ Apoptosis Detection Kit (cat#ab206386, Abcam) Lysotracker™ Red DND-99 (cat# L7528, ThermoFisher) pHrodo™ Red Epidermal Growth Factor (EGF) Conjugate (cat# P35374, ThermoFisher) Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE, cat# T669, ThermoFisher) MitoTracker™ Green FM (cat# M7514, ThermoFisher) CellROX™ Deep Red Reagent (cat# C10422, ThermoFisher) N-Acetyl-L-cysteine (cat# A7250, Sigma-Aldrich) Quercetin (cat# Q4951, Sigma-Aldrich) Dasatinib (cat# CDS023389, Sigma-Aldrich) Polyethylene glycol 400 (PEG400, Fisher Scientific) Annexin V Apoptosis Detection Kit APC (cat# 88-8007-72, ThermoFisher) APC BrdU Flow Kit (cat# 552598, BD Bioscience) InSolution™ Q-VD-OPh, Non-O-methylated (cat# 551476, Millipore) VECTASTAIN® Elite® ABC HRP Kit (Peroxidase, Rabbit IgG; cat# PK-6101, VECTOR Laboratories) Recombinant Mouse TRANCE/RANK L/TNFSF11 Protein (cat# 462-TR, R&D Systems) Recombinant Mouse M-CSF Protein (cat# 461-ML, R&D Systems) alamarBlue™ Cell Viability Reagent (cat# DAL1025, ThermoFisher) Seahorse XF Cell Mito Stress Test Kit (cat# 103015-100, Agilent) ALIZARIN red 2% solution pH 4.2 (cat# 50-317-34, Fisher scientific) Zolendronic acid monohydrate (cat# SML0223, Sigma-Aldrich) OPG-Fc (Amgen Inc.) MC3T3-E1 cells (mouse C57BL/6 calvarial fibroblasts; cat# 99072810, Sigma-Aldrich) ATDC5 cells (mouse 129 teratocarcinoma-derived osteochondral progenitors; cat# 99072806, Sigma-Aldrich)

    Techniques: Fluorescence, Immunofluorescence, Staining, Immunostaining, Marker, Two Tailed Test

    TMEM127 translocates to the lysosomal site of mTORC1 activation upon amino acid stimulation, and limits mTOR recruitment to Rags. (A) Confocal microscopy of HEK293FT cells stably expressing GFP‐TMEM127 deprived of amino acids and re‐exposed to amino acids in identical concentration as those of RPMI media for the designated amount of time. Cells were fixed and stained with endogenous LAMP2 (red) or mTOR (magenta); TMEM127 was represented by green fluorescence; DAPI (blue) stained nuclei, scale bar is 10 μm; inset represents 3-fold zoomed image of the indicated square region. Quantification of colocalization between mTOR and LAMP2, GFP‐TMEM127 and LAMP2, and GFP-TMEM127 and mTOR, from the cells shown in (A) was performed using Mander’s correlation coefficient (CC) as detailed in Materials and Methods; at least 70 cells were quantified from 10 independent fields per condition, from two independent experiments. Graph depicts mean ± SEM of each time point. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test, comparing with corresponding 0 time point, P values are as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Confocal microscopy of Tmem127 WT, KO, or KO expressing MSCV‐TMEM127 (KO + TMEM127) cells deprived of both growth factors overnight and amino acids for 50 min (AA−) and re‐exposed to AA for 15 min (AA+), stained with endogenous LAMP1 (red) and endogenous mTOR (green), DAPI (blue) stains nuclei; scale bar is 10 µm; inset represents 3-fold zoomed image of the indicated square region. Quantification of the colocalization between mTOR and LAMP1 was performed using Mander’s coefficient, as detailed in Materials and Methods. At least 60 cells were quantified from 10 independent fields per condition, from three independent experiments. Statistical analysis was performed using one-way ANOVA test with Tukey’s post-test, P values are as follows: *P < 0.05, **P < 0.01, ***P < 0.0001, n.s., not significant. (C) HA IP of HEK293T expressing HA‐GST‐RagD and Flag-TMEM127 or EV as described in Materials and Methods and probed with mTOR, HA, GFP; corresponding WCLs are shown on the left and β-actin controls for loading; quantification from three biological replicates was performed using Image J and is shown below the figure; graph depicts mean ± SEM. Statistical analysis was performed using paired, two-tailed, Student’s t-test, P-value as indicated. (D) HA IP of HEK293T expressing HA‐GST-RagD and GFP‐TMEM127 or EV and probed with Raptor, HA, TMEM127; corresponding WCLs are shown on the left and β-actin controls for loading; quantification from three biological replicate experiments was performed using Image J and is shown below the figure; graph depicts mean ± SEM. Statistical analysis was performed using paired, two-tailed, Student’s t-test, P-value as indicated. (E) HEK293FT cells stably expressing MSCV-EV or Flag-TMEM127 were transfected with constructs expressing HA‐GST‐RagB/RagD mutant heterodimers, either as HA‐GST‐RagBGTP/RagDGDP (RagACTIVE) or HA‐GST‐RagBGDP/RagDGTP (RagINACTIVE) were starved of amino acids (AA−), followed by AA stimulation for 15 min (AA+) as described in Materials and Methods. Blots were probed with phosphorylated S6 (p‐S6 S240/244), total S6 (T‐S6), HA, TMEM127 and β‐actin antibodies; quantification from three biological replicate experiments was performed using Image J and results are shown below the figure; graphs depict mean ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni’s post-test, *P <0.05, other comparisons, non-significant.

    Journal: Human Molecular Genetics

    Article Title: The TMEM127 human tumor suppressor is a component of the mTORC1 lysosomal nutrient-sensing complex

    doi: 10.1093/hmg/ddy095

    Figure Lengend Snippet: TMEM127 translocates to the lysosomal site of mTORC1 activation upon amino acid stimulation, and limits mTOR recruitment to Rags. (A) Confocal microscopy of HEK293FT cells stably expressing GFP‐TMEM127 deprived of amino acids and re‐exposed to amino acids in identical concentration as those of RPMI media for the designated amount of time. Cells were fixed and stained with endogenous LAMP2 (red) or mTOR (magenta); TMEM127 was represented by green fluorescence; DAPI (blue) stained nuclei, scale bar is 10 μm; inset represents 3-fold zoomed image of the indicated square region. Quantification of colocalization between mTOR and LAMP2, GFP‐TMEM127 and LAMP2, and GFP-TMEM127 and mTOR, from the cells shown in (A) was performed using Mander’s correlation coefficient (CC) as detailed in Materials and Methods; at least 70 cells were quantified from 10 independent fields per condition, from two independent experiments. Graph depicts mean ± SEM of each time point. Statistical analysis was performed using one-way ANOVA with Tukey’s post-test, comparing with corresponding 0 time point, P values are as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (B) Confocal microscopy of Tmem127 WT, KO, or KO expressing MSCV‐TMEM127 (KO + TMEM127) cells deprived of both growth factors overnight and amino acids for 50 min (AA−) and re‐exposed to AA for 15 min (AA+), stained with endogenous LAMP1 (red) and endogenous mTOR (green), DAPI (blue) stains nuclei; scale bar is 10 µm; inset represents 3-fold zoomed image of the indicated square region. Quantification of the colocalization between mTOR and LAMP1 was performed using Mander’s coefficient, as detailed in Materials and Methods. At least 60 cells were quantified from 10 independent fields per condition, from three independent experiments. Statistical analysis was performed using one-way ANOVA test with Tukey’s post-test, P values are as follows: *P < 0.05, **P < 0.01, ***P < 0.0001, n.s., not significant. (C) HA IP of HEK293T expressing HA‐GST‐RagD and Flag-TMEM127 or EV as described in Materials and Methods and probed with mTOR, HA, GFP; corresponding WCLs are shown on the left and β-actin controls for loading; quantification from three biological replicates was performed using Image J and is shown below the figure; graph depicts mean ± SEM. Statistical analysis was performed using paired, two-tailed, Student’s t-test, P-value as indicated. (D) HA IP of HEK293T expressing HA‐GST-RagD and GFP‐TMEM127 or EV and probed with Raptor, HA, TMEM127; corresponding WCLs are shown on the left and β-actin controls for loading; quantification from three biological replicate experiments was performed using Image J and is shown below the figure; graph depicts mean ± SEM. Statistical analysis was performed using paired, two-tailed, Student’s t-test, P-value as indicated. (E) HEK293FT cells stably expressing MSCV-EV or Flag-TMEM127 were transfected with constructs expressing HA‐GST‐RagB/RagD mutant heterodimers, either as HA‐GST‐RagBGTP/RagDGDP (RagACTIVE) or HA‐GST‐RagBGDP/RagDGTP (RagINACTIVE) were starved of amino acids (AA−), followed by AA stimulation for 15 min (AA+) as described in Materials and Methods. Blots were probed with phosphorylated S6 (p‐S6 S240/244), total S6 (T‐S6), HA, TMEM127 and β‐actin antibodies; quantification from three biological replicate experiments was performed using Image J and results are shown below the figure; graphs depict mean ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni’s post-test, *P <0.05, other comparisons, non-significant.

    Article Snippet: Rat Anti-Mouse CD107a (LAMP1) antibody (#553792) was obtained from BD Biosciences.

    Techniques: Activation Assay, Confocal Microscopy, Stable Transfection, Expressing, Concentration Assay, Staining, Fluorescence, Two Tailed Test, Transfection, Construct, Mutagenesis

    TMEM127 interacts with and affects ragulator complex abundance. (A) Ctrl and two independent clones of TMEM127 KO HEK293FT cells generated by CRISPR-Cas9 (KO1, KO2) were transduced with lentivirus carrying either pLKO EV or pLKO-shLAMTOR1 (shL1) construct. Cells were stimulated with amino acids for 15 min after a 2-h amino acid starvation, as described in Figure 1, 72 h after transduction. Blots were probed with antibodies for phosphorylated S6 kinase (p‐S6K T389) and S6 (p‐S6 S240/244), their corresponding total protein (T‐S6K and T‐S6, respectively), LAMTOR1, TMEM127 and β‐actin (loading); three biological replicates were performed. (B) Flag IP of HEK293T cells expressing Flag‐LAMTOR1 or Flag-EV and probed for endogenous TMEM127. Corresponding WCLs were probed with Flag, TMEM127 and β-actin; three biological replicates were performed. (C) HA IP of HEK293T cells expressing HA-TMEM127 or HA-EV and probed for endogenous LAMTOR1. Corresponding WCLs were probed with HA, LAMTOR1 and β-actin antibodies; three biological replicates were performed. (D) Endogenous TMEM127 IP in HEK293T cells using a polyclonal TMEM127 antibody and probed for endogenous LAMTOR1; IgG was used as a negative control, corresponding WCLs are shown. (E) Flag IP of HEK293T cells expressing Flag-TMEM127 and HA-LAMTOR1 WT or HA-LAMTOR1 G2A mutant and probed for HA and Flag; corresponding WCLs are shown and β-actin controls for loading; three biological replicates were performed. (F) Flag IP of HEK293T cells expressing Flag-TMEM127 and probed for endogenous LAMP1 and TMEM127 (both Flag-TMEM127 and endogenous TMEM127 bands are seen on WCLs, shown on the left, β-actin controls for loading. (G) Confocal microscopy of GFP‐TMEM127 in HEK293T control (LAMTOR1 WT) and LAMTOR1 KO cells; scar bar: 10 μm. (H) Fractionated lysates from Ctrl, two independent clones of TMEM127 KO (1 and 2) and LAMTOR1 KO HEK293FT cells showing membrane fraction containing lysosomes (Lyso), cytosolic fractions (Cyto) and WCLs, probed for TMEM127, LAMTOR1, LAMTOR2 and LAMTOR4, and RagC. Tubulin and LAMP2 were used as cytosolic and lysosomal markers, respectively; three biological replicates were performed. (I) WCLs of WT and Tmem127 KO MEFs and probed for endogenous LAMTOR1, LAMTOR2, TMEM127, β-actin controls for loading; three biological replicates were performed. (J) Western blot of HEK293T cell lysates expressing increasing amounts of GFP‐TMEM127 (0–500 ng) and probed for endogenous LAMTOR1, LAMTOR4, RagC, LAMP1 and TMEM127, β‐actin is a loading control; three biological replicates were performed (quantification shown in Supplementary Material, Fig. S3G). (K) WCLs of three pheochromocytomas carrying distinct truncating TMEM127 mutations (MUT samples #2, 3, 4) and four pheochromocytomas with WT TMEM127 sequence (WT samples #1, 5, 6, 7) probed with TMEM127, LAMTOR1, LAMTOR2, RagA, RagB, ATP6v0d1 and β-actin, as loading control. Note the absence of detectable WT or truncated TMEM127 protein in mutant lanes; one of two western blots run with the same panel of samples is shown. (L) Flag IP of HEK293T cells expressing Flag-LAMTOR1 and transfected with the indicated amount of HA-TMEM127-WT or HA-TMEM127 532DupT mutant construct, probed for HA and Flag; corresponding WCLs are shown on the left and β-actin controls for loading. Three biological replicates were performed.

    Journal: Human Molecular Genetics

    Article Title: The TMEM127 human tumor suppressor is a component of the mTORC1 lysosomal nutrient-sensing complex

    doi: 10.1093/hmg/ddy095

    Figure Lengend Snippet: TMEM127 interacts with and affects ragulator complex abundance. (A) Ctrl and two independent clones of TMEM127 KO HEK293FT cells generated by CRISPR-Cas9 (KO1, KO2) were transduced with lentivirus carrying either pLKO EV or pLKO-shLAMTOR1 (shL1) construct. Cells were stimulated with amino acids for 15 min after a 2-h amino acid starvation, as described in Figure 1, 72 h after transduction. Blots were probed with antibodies for phosphorylated S6 kinase (p‐S6K T389) and S6 (p‐S6 S240/244), their corresponding total protein (T‐S6K and T‐S6, respectively), LAMTOR1, TMEM127 and β‐actin (loading); three biological replicates were performed. (B) Flag IP of HEK293T cells expressing Flag‐LAMTOR1 or Flag-EV and probed for endogenous TMEM127. Corresponding WCLs were probed with Flag, TMEM127 and β-actin; three biological replicates were performed. (C) HA IP of HEK293T cells expressing HA-TMEM127 or HA-EV and probed for endogenous LAMTOR1. Corresponding WCLs were probed with HA, LAMTOR1 and β-actin antibodies; three biological replicates were performed. (D) Endogenous TMEM127 IP in HEK293T cells using a polyclonal TMEM127 antibody and probed for endogenous LAMTOR1; IgG was used as a negative control, corresponding WCLs are shown. (E) Flag IP of HEK293T cells expressing Flag-TMEM127 and HA-LAMTOR1 WT or HA-LAMTOR1 G2A mutant and probed for HA and Flag; corresponding WCLs are shown and β-actin controls for loading; three biological replicates were performed. (F) Flag IP of HEK293T cells expressing Flag-TMEM127 and probed for endogenous LAMP1 and TMEM127 (both Flag-TMEM127 and endogenous TMEM127 bands are seen on WCLs, shown on the left, β-actin controls for loading. (G) Confocal microscopy of GFP‐TMEM127 in HEK293T control (LAMTOR1 WT) and LAMTOR1 KO cells; scar bar: 10 μm. (H) Fractionated lysates from Ctrl, two independent clones of TMEM127 KO (1 and 2) and LAMTOR1 KO HEK293FT cells showing membrane fraction containing lysosomes (Lyso), cytosolic fractions (Cyto) and WCLs, probed for TMEM127, LAMTOR1, LAMTOR2 and LAMTOR4, and RagC. Tubulin and LAMP2 were used as cytosolic and lysosomal markers, respectively; three biological replicates were performed. (I) WCLs of WT and Tmem127 KO MEFs and probed for endogenous LAMTOR1, LAMTOR2, TMEM127, β-actin controls for loading; three biological replicates were performed. (J) Western blot of HEK293T cell lysates expressing increasing amounts of GFP‐TMEM127 (0–500 ng) and probed for endogenous LAMTOR1, LAMTOR4, RagC, LAMP1 and TMEM127, β‐actin is a loading control; three biological replicates were performed (quantification shown in Supplementary Material, Fig. S3G). (K) WCLs of three pheochromocytomas carrying distinct truncating TMEM127 mutations (MUT samples #2, 3, 4) and four pheochromocytomas with WT TMEM127 sequence (WT samples #1, 5, 6, 7) probed with TMEM127, LAMTOR1, LAMTOR2, RagA, RagB, ATP6v0d1 and β-actin, as loading control. Note the absence of detectable WT or truncated TMEM127 protein in mutant lanes; one of two western blots run with the same panel of samples is shown. (L) Flag IP of HEK293T cells expressing Flag-LAMTOR1 and transfected with the indicated amount of HA-TMEM127-WT or HA-TMEM127 532DupT mutant construct, probed for HA and Flag; corresponding WCLs are shown on the left and β-actin controls for loading. Three biological replicates were performed.

    Article Snippet: Rat Anti-Mouse CD107a (LAMP1) antibody (#553792) was obtained from BD Biosciences.

    Techniques: Clone Assay, Generated, CRISPR, Transduction, Construct, Expressing, Negative Control, Mutagenesis, Confocal Microscopy, Western Blot, Sequencing, Transfection

    TMEM127 interacts with vATPase and Rag GTPases. (A) Flag IP of HEK293T cells expressing Flag‐TMEM127 and probed for endogenous vATPase subunits (ATP6V0d1 and ATP6V1A1), LAMP1 and Flag; IgG was used as negative control; corresponding WCLs are shown on the left; three biological replicates were performed. (B) IP of endogenous TMEM127 in HEK293T cells using a polyclonal TMEM127 antibody and probed for endogenous ATP6V0d1; IgG was used as a negative control, *indicates a non-specific band, corresponding WCLs are shown on the left. (C) HA IP of Ctrl and LAMTOR1 KO HEK293FT cells expressing HA-TMEM127 or HA-EV and probed for endogenous ATP6V0d1, LAMTOR1 and HA; corresponding WCLs are shown on the left, β-actin controls for loading; quantification was performed with Image J; graph depicts mean ±SEM. Statistics was determined from three biological replicate experiments using paired, two-tailed, Student’s t test, n.s., nonsignificant. (D) Flag IP of HEK293FT cells stably expressing Flag-TMEM127 treated with DMSO or ConA 2 μm for 2 h and probed for ATP6V0d1 and Flag. Corresponding WCLs are shown on the left, LC3 was used as control for ConA treatment and β-actin controls for loading; quantification was performed with Image J; graph depicts mean ±SEM. Statistics was determined from three biological replicate experiments using paired, two-tailed, Student’s t-test, *P < 0.05. (E) HA IP of HEK293T cells expressing GFP‐TMEM127 and HA-GST-RagD probed for GFP, HA; corresponding WCLs are shown on the left; β-actin controls for loading; three biological replicates were performed. (F) HA IP of HEK293T cells expressing HA‐TMEM127 or HA-EV and probed for endogenous RagA or HA; corresponding WCLs are shown on the left and β-actin controls for loading; three biological replicates were performed.

    Journal: Human Molecular Genetics

    Article Title: The TMEM127 human tumor suppressor is a component of the mTORC1 lysosomal nutrient-sensing complex

    doi: 10.1093/hmg/ddy095

    Figure Lengend Snippet: TMEM127 interacts with vATPase and Rag GTPases. (A) Flag IP of HEK293T cells expressing Flag‐TMEM127 and probed for endogenous vATPase subunits (ATP6V0d1 and ATP6V1A1), LAMP1 and Flag; IgG was used as negative control; corresponding WCLs are shown on the left; three biological replicates were performed. (B) IP of endogenous TMEM127 in HEK293T cells using a polyclonal TMEM127 antibody and probed for endogenous ATP6V0d1; IgG was used as a negative control, *indicates a non-specific band, corresponding WCLs are shown on the left. (C) HA IP of Ctrl and LAMTOR1 KO HEK293FT cells expressing HA-TMEM127 or HA-EV and probed for endogenous ATP6V0d1, LAMTOR1 and HA; corresponding WCLs are shown on the left, β-actin controls for loading; quantification was performed with Image J; graph depicts mean ±SEM. Statistics was determined from three biological replicate experiments using paired, two-tailed, Student’s t test, n.s., nonsignificant. (D) Flag IP of HEK293FT cells stably expressing Flag-TMEM127 treated with DMSO or ConA 2 μm for 2 h and probed for ATP6V0d1 and Flag. Corresponding WCLs are shown on the left, LC3 was used as control for ConA treatment and β-actin controls for loading; quantification was performed with Image J; graph depicts mean ±SEM. Statistics was determined from three biological replicate experiments using paired, two-tailed, Student’s t-test, *P < 0.05. (E) HA IP of HEK293T cells expressing GFP‐TMEM127 and HA-GST-RagD probed for GFP, HA; corresponding WCLs are shown on the left; β-actin controls for loading; three biological replicates were performed. (F) HA IP of HEK293T cells expressing HA‐TMEM127 or HA-EV and probed for endogenous RagA or HA; corresponding WCLs are shown on the left and β-actin controls for loading; three biological replicates were performed.

    Article Snippet: Rat Anti-Mouse CD107a (LAMP1) antibody (#553792) was obtained from BD Biosciences.

    Techniques: Expressing, Negative Control, Two Tailed Test, Stable Transfection

    Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .

    Journal: The Journal of Cell Biology

    Article Title: The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

    doi: 10.1083/jcb.201310043

    Figure Lengend Snippet: Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8 . (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14 −/− ;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9 . (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14 f/− MEFs treated with control RNAi (mean of FA length in control p14 f/− MEFs treated with control RNAi was taken as 100%). See also Table S1 . The graph on the right shows the migration speed of p14 −/− ;p14-GFP MEFs transfected with control ( n = 26) and Arl8b siRNA ( n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10 .

    Article Snippet: The mouse monoclonal anti-Paxillin antibody was bought from EMD Millipore, and the rat anti-LAMP1 (CD107a) antibody was purchased from Merck.

    Techniques: Expressing, Transfection, Migration, Wound Healing Assay

    Absence of p14–MP1 or blockage of Arl8b-dependent late endosomal transport causes IQGAP1 accumulation in FAs. (A) IF: anti-Paxillin and anti-IQGAP1 antibodies. IQGAP1 localizes to the leading edge in p14 f/− (white arrows) and colocalizes with Paxillin in FAs in p14 −/− MEFs (red arrows). (B) IF: anti-Paxillin and anti-IQGAP1 antibodies. Shown are different time points during spreading of p14 f/− and p14 −/− MEFs. White arrows indicate accumulation of IQGAP1 at the leading edge in control MEFs. Red arrows point at IQGAP1 localization at Paxillin-positive FAs in control and p14 −/− MEFs. (C) IF: anti-LAMP1 (green), anti-IQGAP1 (red) antibodies, and Hoechst (blue). IQGAP1 localizes to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) and localizes to FAs upon Arl8b depletion (red arrows). (D) p14 f/− MEFs treated as in C. IF: anti-Paxillin and anti-IQGAP1 antibodies. Note IQGAP1 localization to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) versus colocalization of IQGAP1 and Paxillin in FAs upon ARl8b depletion (red arrows).

    Journal: The Journal of Cell Biology

    Article Title: The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

    doi: 10.1083/jcb.201310043

    Figure Lengend Snippet: Absence of p14–MP1 or blockage of Arl8b-dependent late endosomal transport causes IQGAP1 accumulation in FAs. (A) IF: anti-Paxillin and anti-IQGAP1 antibodies. IQGAP1 localizes to the leading edge in p14 f/− (white arrows) and colocalizes with Paxillin in FAs in p14 −/− MEFs (red arrows). (B) IF: anti-Paxillin and anti-IQGAP1 antibodies. Shown are different time points during spreading of p14 f/− and p14 −/− MEFs. White arrows indicate accumulation of IQGAP1 at the leading edge in control MEFs. Red arrows point at IQGAP1 localization at Paxillin-positive FAs in control and p14 −/− MEFs. (C) IF: anti-LAMP1 (green), anti-IQGAP1 (red) antibodies, and Hoechst (blue). IQGAP1 localizes to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) and localizes to FAs upon Arl8b depletion (red arrows). (D) p14 f/− MEFs treated as in C. IF: anti-Paxillin and anti-IQGAP1 antibodies. Note IQGAP1 localization to the leading edge in p14 f/− MEFs treated with control RNAi (white arrows) versus colocalization of IQGAP1 and Paxillin in FAs upon ARl8b depletion (red arrows).

    Article Snippet: The mouse monoclonal anti-Paxillin antibody was bought from EMD Millipore, and the rat anti-LAMP1 (CD107a) antibody was purchased from Merck.

    Techniques: