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lc3b  (OriGene)


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    OriGene lc3b
    Lc3b, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/dynamin+2/Dynamin+2+(DNM2)+(NM_001005360)+Human+Recombinant+Protein/pm32315611-331-2-7
    Average 90 stars, based on 1 article reviews
    lc3b - by Bioz Stars, 2026-10
    90/100 stars

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    Related Articles

    Binding Assay:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Purification:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Incubation:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Recombinant:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti AP2 Thermofisher Cat#(MA1-064) AB_2258307 rabbit anti-ATG3 ABCAM Cat#(ab108251) AB_10865145 rabbit anti-ATG9A ABCAM Cat#(ab108338) AB_10863880 rabbit anti-ATG9A Eurogentec rabbit 1963 (This paper) rabbit anti-ATG16L1 Cell Signaling Cat#(D6D5) AB_10950320 mouse anti-Cathepsin D ABCAM Cat#(ab6313) AB_305416 rabbit anti DNM2 ABCAM Cat#(ab65556) AB_1603781 mouse anti-Flag SIGMA Cat#(F1804) AB_262044 rabbit anti-GABARAP GENERON Cat#(AP1821A) AB_2278762 rabbit anti-GABARAP-L1 Proteintech Cat#(110101) AB_2294415 GABARAP-L2 MBL Cat#(PM038) AB_843130 mouse anti-GAPDH ABCAM Cat#(ab8245) AB_2107448 rabbit anti-GFP Invitrogen Cat#(A6455) AB_221570 mouse anti-HA Biolegend Cat#(16B12) AB_10063630 rabbit anti-ITSN1 Millipore Cat#(ABN1378) mouse anti-LC3B Nanotools Cat#(0231-100/LC3-5F10) AB_2722733 rabbit anti-LC3B ABCAM Cat#(ab192890) rabbit anti-P62 MBL Cat#(PM045) AB_1279301 mouse anti-Myc tag ABCAM ab18185 AB_444307 rabbit anti-RAB11A ABCAM Cat# (ab128913) AB_11140633 Chemicals, Peptides, and Recombinant Proteins Ferrofluid EMG508 Megatech ltd. Cat# (TSD-06-24-11-1) Bafilomycin A1 Enzo Cat# (BML-CM110) SMER28 Tocris Cat# (4297) Rapamycin LC Laboratories Cat# (R-5000) HaloTag TMR ligand Promega Cat# (G825A) HaloTag Alexa 488 ligand Promega Cat# (G100A) Seahorse XF Plasma membrane permeabilizer Agilent Technologies Cat# (102504-100) DNM2 recombinant protein Origene, Cat# (TP323585) LC3 recombinant protein This paper N/A (Continued on next page) Developmental Cell 53, 154–168.e1–e6, April 20, 2020 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays GFP-TRAP Chromotek Cat# (Gtma-20) Myc-TRAP Chromotek Cat# (ytma-20) Anti-HA Magnetic Beads Pierce Cat# (88836) Experimental Models: Cell Lines U2OS Halotag-LC3 This paper N/A HeLa DNM2-PEGFP WT This paper N/A HeLa DNM2-PEGFP R465W This paper N/A HeLa DNM2-PEGFP W525L This paper N/A RFP-GFP tandem fluorescent-tagged LC3 Ravikumar et al., 2010 N/A Mouse embryonic fibroblasts DNM2R465W Durieux et al., 2010 N/A Mouse embryonic fibroblasts WT Durieux et al., 2010 N/A Patient-derived fibroblasts DNM2 R465W Trochet et al., 2018 N/A Control human fibroblasts C1 Trochet et al., 2018 N/A Control human fibroblasts 711 Coriell Institute GM04711 Control human fibroblasts 729 Coriell Institute GM04729 Control human fibroblasts 865 Coriell Institute GM04865 Oligonucleotides (primers) Primer for DNM2 R465W: TCA CCA CCT ACA TCT GGG AGC GAG AAG GG This paper N/A Primer for DNM2 W525L: TGA TCC GCA GGG GCT TGT TGA CCA TCA ACA AC This paper N/A Primer for LC3 K51A: CCG TCC TGG ACA AGA CCG CGT TCC TTG TAC CTG ATC This paper N/A Primer for LC3 R70A: CAT CAA GAT AAT TAG AAG GGC CCT GCA GCT CAA TGC TAA C This paper N/A For siRNA sequences, see Table S1 Recombinant DNA pEGFP-RAB11A Puri et al., 2013 N/A mCherry-RAB11A Puri et al., 2018a N/A pEGFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A pECFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A RFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A LC3-PGEX-6P-1 Mineyuki Mizuguchi (Toyama University, Japan) N/A DNM2-WT-PEGFP Mark McNiven (Mayo Clinic Minnesota, USA) N/A HA -DNM2-WT Addgene Cat# (34684) Flag-ITSN1-short Addgene Cat# (47392) pEGFP-ITSN1 short Addgene Cat# (47394) Flag-empty vector SIGMA Cat# (E4901) Myc-SNX18-WT Anne Simonsen (University of Oslo, Norway) N/A Myc-SNX18- W38F Anne Simonsen (University of Oslo, Norway) N/A httQ74-HA Korolchuk et al., 2009 N/A Software and Algorithms Prism 7 GraphPad N/A ZEN Black Carl Zeiss Microscopy N/A Volocity PerkinElmer N/A Image Studio Lite LI-COR, Inc N/A ImageJ National Institute of Health, USA N/A e2 Developmental Cell 53, 154–168.e1–e6, April 20, 2020

    Immunoprecipitation:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Western Blot:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: PreScission Protease (GE Healthcare, 27-0843-01) was added at 100 units/ml in a 2-bed volume of PreScission Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly prepared with 1 mM DTT and cleavage was performed overnight at 4 C. Cleaved protein was eluted and stored at -80 C. .. Binding of LC3B (purified fromE.Coli) to DNM2 (Origene, TP323585) was performed by incubation of recombinant proteins at 250C for 1 h, followed by immunoprecipitation with rabbit anti-DNM2 antibody (ABCAM, ab65556-1:700) and processed for western blot analysis. ..

    Clinical Proteomics:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti AP2 Thermofisher Cat#(MA1-064) AB_2258307 rabbit anti-ATG3 ABCAM Cat#(ab108251) AB_10865145 rabbit anti-ATG9A ABCAM Cat#(ab108338) AB_10863880 rabbit anti-ATG9A Eurogentec rabbit 1963 (This paper) rabbit anti-ATG16L1 Cell Signaling Cat#(D6D5) AB_10950320 mouse anti-Cathepsin D ABCAM Cat#(ab6313) AB_305416 rabbit anti DNM2 ABCAM Cat#(ab65556) AB_1603781 mouse anti-Flag SIGMA Cat#(F1804) AB_262044 rabbit anti-GABARAP GENERON Cat#(AP1821A) AB_2278762 rabbit anti-GABARAP-L1 Proteintech Cat#(110101) AB_2294415 GABARAP-L2 MBL Cat#(PM038) AB_843130 mouse anti-GAPDH ABCAM Cat#(ab8245) AB_2107448 rabbit anti-GFP Invitrogen Cat#(A6455) AB_221570 mouse anti-HA Biolegend Cat#(16B12) AB_10063630 rabbit anti-ITSN1 Millipore Cat#(ABN1378) mouse anti-LC3B Nanotools Cat#(0231-100/LC3-5F10) AB_2722733 rabbit anti-LC3B ABCAM Cat#(ab192890) rabbit anti-P62 MBL Cat#(PM045) AB_1279301 mouse anti-Myc tag ABCAM ab18185 AB_444307 rabbit anti-RAB11A ABCAM Cat# (ab128913) AB_11140633 Chemicals, Peptides, and Recombinant Proteins Ferrofluid EMG508 Megatech ltd. Cat# (TSD-06-24-11-1) Bafilomycin A1 Enzo Cat# (BML-CM110) SMER28 Tocris Cat# (4297) Rapamycin LC Laboratories Cat# (R-5000) HaloTag TMR ligand Promega Cat# (G825A) HaloTag Alexa 488 ligand Promega Cat# (G100A) Seahorse XF Plasma membrane permeabilizer Agilent Technologies Cat# (102504-100) DNM2 recombinant protein Origene, Cat# (TP323585) LC3 recombinant protein This paper N/A (Continued on next page) Developmental Cell 53, 154–168.e1–e6, April 20, 2020 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays GFP-TRAP Chromotek Cat# (Gtma-20) Myc-TRAP Chromotek Cat# (ytma-20) Anti-HA Magnetic Beads Pierce Cat# (88836) Experimental Models: Cell Lines U2OS Halotag-LC3 This paper N/A HeLa DNM2-PEGFP WT This paper N/A HeLa DNM2-PEGFP R465W This paper N/A HeLa DNM2-PEGFP W525L This paper N/A RFP-GFP tandem fluorescent-tagged LC3 Ravikumar et al., 2010 N/A Mouse embryonic fibroblasts DNM2R465W Durieux et al., 2010 N/A Mouse embryonic fibroblasts WT Durieux et al., 2010 N/A Patient-derived fibroblasts DNM2 R465W Trochet et al., 2018 N/A Control human fibroblasts C1 Trochet et al., 2018 N/A Control human fibroblasts 711 Coriell Institute GM04711 Control human fibroblasts 729 Coriell Institute GM04729 Control human fibroblasts 865 Coriell Institute GM04865 Oligonucleotides (primers) Primer for DNM2 R465W: TCA CCA CCT ACA TCT GGG AGC GAG AAG GG This paper N/A Primer for DNM2 W525L: TGA TCC GCA GGG GCT TGT TGA CCA TCA ACA AC This paper N/A Primer for LC3 K51A: CCG TCC TGG ACA AGA CCG CGT TCC TTG TAC CTG ATC This paper N/A Primer for LC3 R70A: CAT CAA GAT AAT TAG AAG GGC CCT GCA GCT CAA TGC TAA C This paper N/A For siRNA sequences, see Table S1 Recombinant DNA pEGFP-RAB11A Puri et al., 2013 N/A mCherry-RAB11A Puri et al., 2018a N/A pEGFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A pECFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A RFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A LC3-PGEX-6P-1 Mineyuki Mizuguchi (Toyama University, Japan) N/A DNM2-WT-PEGFP Mark McNiven (Mayo Clinic Minnesota, USA) N/A HA -DNM2-WT Addgene Cat# (34684) Flag-ITSN1-short Addgene Cat# (47392) pEGFP-ITSN1 short Addgene Cat# (47394) Flag-empty vector SIGMA Cat# (E4901) Myc-SNX18-WT Anne Simonsen (University of Oslo, Norway) N/A Myc-SNX18- W38F Anne Simonsen (University of Oslo, Norway) N/A httQ74-HA Korolchuk et al., 2009 N/A Software and Algorithms Prism 7 GraphPad N/A ZEN Black Carl Zeiss Microscopy N/A Volocity PerkinElmer N/A Image Studio Lite LI-COR, Inc N/A ImageJ National Institute of Health, USA N/A e2 Developmental Cell 53, 154–168.e1–e6, April 20, 2020

    Membrane:

    Article Title: A DNM2 Centronuclear Myopathy Mutation Reveals a Link between Recycling Endosome Scission and Autophagy.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies mouse anti AP2 Thermofisher Cat#(MA1-064) AB_2258307 rabbit anti-ATG3 ABCAM Cat#(ab108251) AB_10865145 rabbit anti-ATG9A ABCAM Cat#(ab108338) AB_10863880 rabbit anti-ATG9A Eurogentec rabbit 1963 (This paper) rabbit anti-ATG16L1 Cell Signaling Cat#(D6D5) AB_10950320 mouse anti-Cathepsin D ABCAM Cat#(ab6313) AB_305416 rabbit anti DNM2 ABCAM Cat#(ab65556) AB_1603781 mouse anti-Flag SIGMA Cat#(F1804) AB_262044 rabbit anti-GABARAP GENERON Cat#(AP1821A) AB_2278762 rabbit anti-GABARAP-L1 Proteintech Cat#(110101) AB_2294415 GABARAP-L2 MBL Cat#(PM038) AB_843130 mouse anti-GAPDH ABCAM Cat#(ab8245) AB_2107448 rabbit anti-GFP Invitrogen Cat#(A6455) AB_221570 mouse anti-HA Biolegend Cat#(16B12) AB_10063630 rabbit anti-ITSN1 Millipore Cat#(ABN1378) mouse anti-LC3B Nanotools Cat#(0231-100/LC3-5F10) AB_2722733 rabbit anti-LC3B ABCAM Cat#(ab192890) rabbit anti-P62 MBL Cat#(PM045) AB_1279301 mouse anti-Myc tag ABCAM ab18185 AB_444307 rabbit anti-RAB11A ABCAM Cat# (ab128913) AB_11140633 Chemicals, Peptides, and Recombinant Proteins Ferrofluid EMG508 Megatech ltd. Cat# (TSD-06-24-11-1) Bafilomycin A1 Enzo Cat# (BML-CM110) SMER28 Tocris Cat# (4297) Rapamycin LC Laboratories Cat# (R-5000) HaloTag TMR ligand Promega Cat# (G825A) HaloTag Alexa 488 ligand Promega Cat# (G100A) Seahorse XF Plasma membrane permeabilizer Agilent Technologies Cat# (102504-100) DNM2 recombinant protein Origene, Cat# (TP323585) LC3 recombinant protein This paper N/A (Continued on next page) Developmental Cell 53, 154–168.e1–e6, April 20, 2020 e1 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays GFP-TRAP Chromotek Cat# (Gtma-20) Myc-TRAP Chromotek Cat# (ytma-20) Anti-HA Magnetic Beads Pierce Cat# (88836) Experimental Models: Cell Lines U2OS Halotag-LC3 This paper N/A HeLa DNM2-PEGFP WT This paper N/A HeLa DNM2-PEGFP R465W This paper N/A HeLa DNM2-PEGFP W525L This paper N/A RFP-GFP tandem fluorescent-tagged LC3 Ravikumar et al., 2010 N/A Mouse embryonic fibroblasts DNM2R465W Durieux et al., 2010 N/A Mouse embryonic fibroblasts WT Durieux et al., 2010 N/A Patient-derived fibroblasts DNM2 R465W Trochet et al., 2018 N/A Control human fibroblasts C1 Trochet et al., 2018 N/A Control human fibroblasts 711 Coriell Institute GM04711 Control human fibroblasts 729 Coriell Institute GM04729 Control human fibroblasts 865 Coriell Institute GM04865 Oligonucleotides (primers) Primer for DNM2 R465W: TCA CCA CCT ACA TCT GGG AGC GAG AAG GG This paper N/A Primer for DNM2 W525L: TGA TCC GCA GGG GCT TGT TGA CCA TCA ACA AC This paper N/A Primer for LC3 K51A: CCG TCC TGG ACA AGA CCG CGT TCC TTG TAC CTG ATC This paper N/A Primer for LC3 R70A: CAT CAA GAT AAT TAG AAG GGC CCT GCA GCT CAA TGC TAA C This paper N/A For siRNA sequences, see Table S1 Recombinant DNA pEGFP-RAB11A Puri et al., 2013 N/A mCherry-RAB11A Puri et al., 2018a N/A pEGFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A pECFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A RFP-LC3 Tamotsu Yoshimori (Osaka University, Japan) N/A LC3-PGEX-6P-1 Mineyuki Mizuguchi (Toyama University, Japan) N/A DNM2-WT-PEGFP Mark McNiven (Mayo Clinic Minnesota, USA) N/A HA -DNM2-WT Addgene Cat# (34684) Flag-ITSN1-short Addgene Cat# (47392) pEGFP-ITSN1 short Addgene Cat# (47394) Flag-empty vector SIGMA Cat# (E4901) Myc-SNX18-WT Anne Simonsen (University of Oslo, Norway) N/A Myc-SNX18- W38F Anne Simonsen (University of Oslo, Norway) N/A httQ74-HA Korolchuk et al., 2009 N/A Software and Algorithms Prism 7 GraphPad N/A ZEN Black Carl Zeiss Microscopy N/A Volocity PerkinElmer N/A Image Studio Lite LI-COR, Inc N/A ImageJ National Institute of Health, USA N/A e2 Developmental Cell 53, 154–168.e1–e6, April 20, 2020



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    Bethyl anti dynamin 2 antibody
    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 <t>(K44A)-GFP</t> to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.
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    513a t  (Bethyl)
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    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 <t>(K44A)-GFP</t> to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.
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    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 <t>(K44A)-GFP</t> to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.
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    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 <t>(K44A)-GFP</t> to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.
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    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 <t>(K44A)-GFP</t> to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.
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    Image Search Results


    (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

    Journal: bioRxiv

    Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

    doi: 10.64898/2026.01.30.702748

    Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

    Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

    Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis