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antibodies against mth1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against mth1
    A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in <t>FLAG-MTH1-RNF152</t> stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with <t>MTH1</t> and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.
    Antibodies Against Mth1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against mth1/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2718 article reviews
    antibodies against mth1 - by Bioz Stars, 2026-02
    99/100 stars

    Images

    1) Product Images from "LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation"

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    Journal: bioRxiv

    doi: 10.1101/2024.09.08.611923

    A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.
    Figure Legend Snippet: A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.

    Techniques Used: Membrane, Stable Transfection, Expressing, Ubiquitin Proteomics, Control, Western Blot, Quantitative Proteomics

    A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.
    Figure Legend Snippet: A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.

    Techniques Used: Degradation Assay, Stable Transfection, Expressing, Knock-In, Western Blot, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

    A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.
    Figure Legend Snippet: A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.

    Techniques Used: Degradation Assay, Stable Transfection, Expressing, Knock-In, Tube Formation Assay, Immunoprecipitation, Western Blot, Control

    A) Schematic of LYMTAC-induced KRAS relocalization from plasma membrane to lysosome. B) KRAS localization analyzed by confocal microscopy. FLAG-MTH1-RNF152-stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min, and then co-treated with DMSO or 1 µM LYMTAC-4 for 4 h. Cells were fixed, permeabilized, and immunostained with FLAG and LAMP1 antibodies, followed by appropriate secondary fluorophore-conjugated antibodies. Nuclei were stained with Hoechst dye. Data are representative of two independent experiments. C) Confocal images in FLAG-MTH1-RNF152 stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 or 1 µM Bortezomib for 30 min before 4 h co-treatment with DMSO (+BafA1), 1 µM pan-KRAS inhibitor, 1 µM PROTAC (+Bortezomib), or 1 µM LYMTAC-4 (+BafA1). Cells were fixed and nuclei were stained with Hoechst dye. Data are representative of two independent experiments. D) Washout experiment in AsPC-1 cells stably expressing FLAG-MTH1-RNF152. Cells were treated with 100 nM pan-KRASi and 100 nM LYMTAC-4 for 4 h, washed three times with PBS and replaced with fresh media in the absence or presence of 5 µM MTH1-ligand for 48 h. Data are representative of two independent experiments. E) pan-KRASi, PROTAC, and LYMTAC-4 activity in a 5-day cell proliferation assay in AsPC-1 cells stably expressing MTH1-RNF152. Data are representative of four independent experiments, reported as the mean ± S.E.
    Figure Legend Snippet: A) Schematic of LYMTAC-induced KRAS relocalization from plasma membrane to lysosome. B) KRAS localization analyzed by confocal microscopy. FLAG-MTH1-RNF152-stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min, and then co-treated with DMSO or 1 µM LYMTAC-4 for 4 h. Cells were fixed, permeabilized, and immunostained with FLAG and LAMP1 antibodies, followed by appropriate secondary fluorophore-conjugated antibodies. Nuclei were stained with Hoechst dye. Data are representative of two independent experiments. C) Confocal images in FLAG-MTH1-RNF152 stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 or 1 µM Bortezomib for 30 min before 4 h co-treatment with DMSO (+BafA1), 1 µM pan-KRAS inhibitor, 1 µM PROTAC (+Bortezomib), or 1 µM LYMTAC-4 (+BafA1). Cells were fixed and nuclei were stained with Hoechst dye. Data are representative of two independent experiments. D) Washout experiment in AsPC-1 cells stably expressing FLAG-MTH1-RNF152. Cells were treated with 100 nM pan-KRASi and 100 nM LYMTAC-4 for 4 h, washed three times with PBS and replaced with fresh media in the absence or presence of 5 µM MTH1-ligand for 48 h. Data are representative of two independent experiments. E) pan-KRASi, PROTAC, and LYMTAC-4 activity in a 5-day cell proliferation assay in AsPC-1 cells stably expressing MTH1-RNF152. Data are representative of four independent experiments, reported as the mean ± S.E.

    Techniques Used: Clinical Proteomics, Membrane, Confocal Microscopy, Stable Transfection, Expressing, Staining, Activity Assay, Proliferation Assay

    A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.
    Figure Legend Snippet: A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.

    Techniques Used: Membrane, Stable Transfection, Expressing, Control, Western Blot, Degradation Assay, Knock-In, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation



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    A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in <t>FLAG-MTH1-RNF152</t> stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with <t>MTH1</t> and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.
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    A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    doi: 10.1101/2024.09.08.611923

    Figure Lengend Snippet: A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.

    Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

    Techniques: Membrane, Stable Transfection, Expressing, Ubiquitin Proteomics, Control, Western Blot, Quantitative Proteomics

    A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    doi: 10.1101/2024.09.08.611923

    Figure Lengend Snippet: A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.

    Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

    Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Western Blot, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

    A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    doi: 10.1101/2024.09.08.611923

    Figure Lengend Snippet: A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.

    Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

    Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Tube Formation Assay, Immunoprecipitation, Western Blot, Control

    A) Schematic of LYMTAC-induced KRAS relocalization from plasma membrane to lysosome. B) KRAS localization analyzed by confocal microscopy. FLAG-MTH1-RNF152-stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min, and then co-treated with DMSO or 1 µM LYMTAC-4 for 4 h. Cells were fixed, permeabilized, and immunostained with FLAG and LAMP1 antibodies, followed by appropriate secondary fluorophore-conjugated antibodies. Nuclei were stained with Hoechst dye. Data are representative of two independent experiments. C) Confocal images in FLAG-MTH1-RNF152 stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 or 1 µM Bortezomib for 30 min before 4 h co-treatment with DMSO (+BafA1), 1 µM pan-KRAS inhibitor, 1 µM PROTAC (+Bortezomib), or 1 µM LYMTAC-4 (+BafA1). Cells were fixed and nuclei were stained with Hoechst dye. Data are representative of two independent experiments. D) Washout experiment in AsPC-1 cells stably expressing FLAG-MTH1-RNF152. Cells were treated with 100 nM pan-KRASi and 100 nM LYMTAC-4 for 4 h, washed three times with PBS and replaced with fresh media in the absence or presence of 5 µM MTH1-ligand for 48 h. Data are representative of two independent experiments. E) pan-KRASi, PROTAC, and LYMTAC-4 activity in a 5-day cell proliferation assay in AsPC-1 cells stably expressing MTH1-RNF152. Data are representative of four independent experiments, reported as the mean ± S.E.

    Journal: bioRxiv

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    doi: 10.1101/2024.09.08.611923

    Figure Lengend Snippet: A) Schematic of LYMTAC-induced KRAS relocalization from plasma membrane to lysosome. B) KRAS localization analyzed by confocal microscopy. FLAG-MTH1-RNF152-stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 for 30 min, and then co-treated with DMSO or 1 µM LYMTAC-4 for 4 h. Cells were fixed, permeabilized, and immunostained with FLAG and LAMP1 antibodies, followed by appropriate secondary fluorophore-conjugated antibodies. Nuclei were stained with Hoechst dye. Data are representative of two independent experiments. C) Confocal images in FLAG-MTH1-RNF152 stably expressing HEK293T mNeonGreen-KRAS WT cells were pre-treated with 200 nM BafA1 or 1 µM Bortezomib for 30 min before 4 h co-treatment with DMSO (+BafA1), 1 µM pan-KRAS inhibitor, 1 µM PROTAC (+Bortezomib), or 1 µM LYMTAC-4 (+BafA1). Cells were fixed and nuclei were stained with Hoechst dye. Data are representative of two independent experiments. D) Washout experiment in AsPC-1 cells stably expressing FLAG-MTH1-RNF152. Cells were treated with 100 nM pan-KRASi and 100 nM LYMTAC-4 for 4 h, washed three times with PBS and replaced with fresh media in the absence or presence of 5 µM MTH1-ligand for 48 h. Data are representative of two independent experiments. E) pan-KRASi, PROTAC, and LYMTAC-4 activity in a 5-day cell proliferation assay in AsPC-1 cells stably expressing MTH1-RNF152. Data are representative of four independent experiments, reported as the mean ± S.E.

    Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

    Techniques: Clinical Proteomics, Membrane, Confocal Microscopy, Stable Transfection, Expressing, Staining, Activity Assay, Proliferation Assay

    A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

    doi: 10.1101/2024.09.08.611923

    Figure Lengend Snippet: A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.

    Article Snippet: Antibodies against MTH1 (43918), FLAG (14793), Vinculin (13901), Tubulin (2144), pERK (9101), ERK (4695), PTK2 (3285), EPHA2 (6997), HA (3724), LAMP1(15665), and GAPDH (2118) were purchased from Cell Signaling Technology.

    Techniques: Membrane, Stable Transfection, Expressing, Control, Western Blot, Degradation Assay, Knock-In, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation