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torin1  (InvivoGen)


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    InvivoGen torin1
    Torin1, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/pm41723185-392-46-47
    Average 95 stars, based on 38 article reviews
    torin1 - by Bioz Stars, 2026-08
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    torin1  (MedChemExpress)
    97
    MedChemExpress torin1
    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and <t>Torin1</t> treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.
    Torin1, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/pmc13387423-49-0-2
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    torin1 - by Bioz Stars, 2026-08
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    torin1  (InvivoGen)
    95
    InvivoGen torin1
    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and <t>Torin1</t> treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.
    Torin1, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/pm41723185-392-46-47
    Average 95 stars, based on 1 article reviews
    torin1 - by Bioz Stars, 2026-08
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    torin1  (Tocris)
    96
    Tocris torin1
    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and <t>Torin1</t> treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.
    Torin1, supplied by Tocris, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/10__1016_slash_j__isci__2026__115860-581-15-18
    Average 96 stars, based on 1 article reviews
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    recombinant proteins torin1 tocris bioscience no 4247 torin2 tocris bioscience no 4248 rapamycin fujifilm wako  (Tocris)
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    Tocris recombinant proteins torin1 tocris bioscience no 4247 torin2 tocris bioscience no 4248 rapamycin fujifilm wako
    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and <t>Torin1</t> treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.
    Recombinant Proteins Torin1 Tocris Bioscience No 4247 Torin2 Tocris Bioscience No 4248 Rapamycin Fujifilm Wako, supplied by Tocris, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant proteins torin1 tocris bioscience no 4247 torin2 tocris bioscience no 4248 rapamycin fujifilm wako - by Bioz Stars, 2026-08
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    torin1  (Selleck Chemicals)
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    Selleck Chemicals torin1
    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and <t>Torin1</t> treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.
    Torin1, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/pm41864210-1012-19-20
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    torin1 - by Bioz Stars, 2026-08
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    torin1  (Cell Signaling Technology Inc)
    95
    Cell Signaling Technology Inc torin1
    a. Western blots showing HMGCS1 protein level in asynchronous hTERT-RPE1 cells that stably express doxycycline-inducible UBE2H constructs (WT, AA and DD). Cells were induced with doxycycline (2.5 ng/ml) for 24 h. Quantification represents mean± s.d. from three independent biological replicates. b. Western blots showing HMGCS1 protein levels in asynchronous or mitotically arrested WT cells and knock-in clones. To induce mitotic arrest, cells were treated with nocodazole (600 nM) for 18h, and mitotic cells were collected by shake-off. Quantification represents mean ± s.d. from three independent biological replicates. c. Western blots showing MKLN1 and HMGCS1 protein levels in WT cells and knock-in clones after treatment with <t>torin1</t> (250 nM), R03306 (7.5 µM) and palbociclib (1 µM) for 18h. Quantification of relative MKLN1and HMGCS1 protein levels is shown (bottom). d. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1 cells and HEK293T cells after treatment with torin1 (250 nM) or rapamycin (80 nM) and the proteasome inhibitor MG262 for 1h. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. e. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT and TSC2- - hTERT-RPE1cells and HEK293T cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. f. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT, TSC2- -and TSC2- - + TSC2 hTERT-RPE1cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. g. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phospho-S6 (p-S6) in hTERT-RPE1, Hela and HEK293T cells that were incubated in glucose-depleted medium for 6h. Quantification represents mean± s.d. from the indicated independent biological replicates. h. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phos-pho-S6 (p-S6) in WT cells and knock-in clones that were incubated in amino-acid (AA)-depleted medium for 6h. Statisti-cal significance of differences between groups was determined with one-way ANOVA analysis (a-b,d, f) or two-tailed unpaired Student’s t-test (e, g). *** = p < 0.001. ** = p < 0.01. * = p < 0.05. ns = not statistically significant.
    Torin1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/torin1/Torin+1/bio_rxiv__64898__2026__03__07__710281-272-45-46
    Average 95 stars, based on 1 article reviews
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    (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and Torin1 treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.

    Journal: Cell reports

    Article Title: Mutant KRAS-driven selective mRNA translation reveals mechanisms and therapeutic vulnerabilities in cancer

    doi: 10.1016/j.celrep.2026.117520

    Figure Lengend Snippet: (A and B) TE fold change analysis of common transcripts affected by sotorasib and CR-1-31B shows that mutant KRAS and EIF4A oppositely regulate the translation of these mRNAs. Most of the sotorasib TE down targets are TE upregulated following EIF4A inhibition with CR-1-31B. (C) TE fold change analysis of common translation factors between sotorasib and CR-1-31B reveals the upregulation of all the translation factors following CR-1-31B treatment, except for four proteins that are downregulated by CR-1-31B. (D and E) Relative luciferase activity driven by G-quadruplex (GQ) and GC-rich motifs in response to sotorasib, RMC-7977, or MRTX1133 treatment demonstrates that sotorasib induces GQ and GC-rich mRNA translation ( * p < 0.05 and *** p < 0.001). (F and G) Luciferase reporter assays using the full-length or mutated 5′UTR of EEF1A1 show altered luciferase activity upon CR-1-31B and Torin1 treatment in MiaPaca-2 cells (* p < 0.05 and *** p < 0.001). (H) Combination treatment of CR-1-31B and Torin1 with mutant KRAS inhibitors in a panel of cancer cell lines harboring either G12C or G12D KRAS mutations leads to an additive enhancement of cell death, based on the zero interaction potency (ZIP) model. (I) Summary of our findings shows that the mutant KRAS-dependent mechanism of translational control uncouples the regulation of translation machinery from the regulation of the rest of the mRNA translation. Mutant KRAS-dependent mechanism is distinct from the mTOR and EIF4A-dependent mechanism that depends on longer and structured 5′UTR features and controls the translation of a distinct subset of mRNAs.

    Article Snippet: Torin1 , MedChemExpress , Cat# HY-13003.

    Techniques: Mutagenesis, Inhibition, Luciferase, Activity Assay, Control

    a. Western blots showing HMGCS1 protein level in asynchronous hTERT-RPE1 cells that stably express doxycycline-inducible UBE2H constructs (WT, AA and DD). Cells were induced with doxycycline (2.5 ng/ml) for 24 h. Quantification represents mean± s.d. from three independent biological replicates. b. Western blots showing HMGCS1 protein levels in asynchronous or mitotically arrested WT cells and knock-in clones. To induce mitotic arrest, cells were treated with nocodazole (600 nM) for 18h, and mitotic cells were collected by shake-off. Quantification represents mean ± s.d. from three independent biological replicates. c. Western blots showing MKLN1 and HMGCS1 protein levels in WT cells and knock-in clones after treatment with torin1 (250 nM), R03306 (7.5 µM) and palbociclib (1 µM) for 18h. Quantification of relative MKLN1and HMGCS1 protein levels is shown (bottom). d. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1 cells and HEK293T cells after treatment with torin1 (250 nM) or rapamycin (80 nM) and the proteasome inhibitor MG262 for 1h. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. e. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT and TSC2- - hTERT-RPE1cells and HEK293T cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. f. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT, TSC2- -and TSC2- - + TSC2 hTERT-RPE1cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. g. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phospho-S6 (p-S6) in hTERT-RPE1, Hela and HEK293T cells that were incubated in glucose-depleted medium for 6h. Quantification represents mean± s.d. from the indicated independent biological replicates. h. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phos-pho-S6 (p-S6) in WT cells and knock-in clones that were incubated in amino-acid (AA)-depleted medium for 6h. Statisti-cal significance of differences between groups was determined with one-way ANOVA analysis (a-b,d, f) or two-tailed unpaired Student’s t-test (e, g). *** = p < 0.001. ** = p < 0.01. * = p < 0.05. ns = not statistically significant.

    Journal: bioRxiv

    Article Title: CDK/mTOR–dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent degradation

    doi: 10.64898/2026.03.07.710281

    Figure Lengend Snippet: a. Western blots showing HMGCS1 protein level in asynchronous hTERT-RPE1 cells that stably express doxycycline-inducible UBE2H constructs (WT, AA and DD). Cells were induced with doxycycline (2.5 ng/ml) for 24 h. Quantification represents mean± s.d. from three independent biological replicates. b. Western blots showing HMGCS1 protein levels in asynchronous or mitotically arrested WT cells and knock-in clones. To induce mitotic arrest, cells were treated with nocodazole (600 nM) for 18h, and mitotic cells were collected by shake-off. Quantification represents mean ± s.d. from three independent biological replicates. c. Western blots showing MKLN1 and HMGCS1 protein levels in WT cells and knock-in clones after treatment with torin1 (250 nM), R03306 (7.5 µM) and palbociclib (1 µM) for 18h. Quantification of relative MKLN1and HMGCS1 protein levels is shown (bottom). d. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1 cells and HEK293T cells after treatment with torin1 (250 nM) or rapamycin (80 nM) and the proteasome inhibitor MG262 for 1h. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. e. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT and TSC2- - hTERT-RPE1cells and HEK293T cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. f. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in WT, TSC2- -and TSC2- - + TSC2 hTERT-RPE1cells. Quantification of normalized level of charged UBE2H is shown as mean± s.d. from three independent biological replicates. g. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phospho-S6 (p-S6) in hTERT-RPE1, Hela and HEK293T cells that were incubated in glucose-depleted medium for 6h. Quantification represents mean± s.d. from the indicated independent biological replicates. h. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates and phos-pho-S6 (p-S6) in WT cells and knock-in clones that were incubated in amino-acid (AA)-depleted medium for 6h. Statisti-cal significance of differences between groups was determined with one-way ANOVA analysis (a-b,d, f) or two-tailed unpaired Student’s t-test (e, g). *** = p < 0.001. ** = p < 0.01. * = p < 0.05. ns = not statistically significant.

    Article Snippet: The following chemicals were used: doxycycline hyclate (Sigma Aldrich, D9891), RO3306 (AdipoGen Life Sciences, AGCR13515M), (R)-roscovitine (AdipoGen Life Sciences, AG-CR1-0006-M005), nocodazole (Selleckchem, S2775), (+)-S-trityl-L-cysteine (STLC) (Alfa Aesar, L14384), proTAME (Boston Biochem, I-440), apcin (Enamine, T0506-3874), taxol (Sigma-Aldrich, 33069-62-4), thymidine (Sigma-Aldrich, T9250), palbociclib (LC Laboratories, P-7722), torin1 (Cell Signaling, 14379S), rapamycin (RPI, R64500-0.001), MG-132 (Calbiochem, 474790), MG-262 (Apexbio, A8179-1), cycloheximide (Sigma, C7698), geranylgeranyl alcohol (Cayman Chemical, 13272), farnesyl alcohol (Cayman Chemical, 13268), puromycin (Thermo Scientific, 22742-0100), bafilomycin A1 (RPI, B40500-0.001), PFI-7 (Gift from C. H. Arrowsmith, Structural Genomics Consortium), SiR-DNA (SiR-Hoechst*) (Spirochrome, SC007), Pierce protease inhibitor tablet (Thermo Scientific, A32953), phosphatase inhibitor tablet (Thermo Scientific, A32957).

    Techniques: Western Blot, Stable Transfection, Construct, Knock-In, Clone Assay, Ubiquitin Proteomics, Incubation, Two Tailed Test

    a. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1and HEK293T cells after treatment with torin1 (250 nM) for the indicated time. Quantification represents mean± s.d. from three independent biological replicates. b. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1 cells and HEK293T cells after treatment with rapamycin at the indicated concentrations for 1 h. Quantification of the percentage of ubiquitin-charged UBE2H is shown (bottom). c. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates after treatment with torin1 (250 nM) and rapamycin (80 nM) in WT, Tsc2- - hTERT-RPE1cells. Quantification of the percentage of charged UBE2H is shown (bottom). d. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in HEK293T cells that were incubated in serum-depleted medium for 12 h. Quantification represents mean ± s.d. from three independent biological replicates. Statistical significance of differences between groups was determined with one-way ANOVA analysis. * = p < 0.05. ns = not statistically significant.

    Journal: bioRxiv

    Article Title: CDK/mTOR–dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent degradation

    doi: 10.64898/2026.03.07.710281

    Figure Lengend Snippet: a. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1and HEK293T cells after treatment with torin1 (250 nM) for the indicated time. Quantification represents mean± s.d. from three independent biological replicates. b. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in hTERT-RPE1 cells and HEK293T cells after treatment with rapamycin at the indicated concentrations for 1 h. Quantification of the percentage of ubiquitin-charged UBE2H is shown (bottom). c. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates after treatment with torin1 (250 nM) and rapamycin (80 nM) in WT, Tsc2- - hTERT-RPE1cells. Quantification of the percentage of charged UBE2H is shown (bottom). d. Non-reducing SOS-PAGE analysis of UBE2H thioester-linked ubiquitin conjugates in HEK293T cells that were incubated in serum-depleted medium for 12 h. Quantification represents mean ± s.d. from three independent biological replicates. Statistical significance of differences between groups was determined with one-way ANOVA analysis. * = p < 0.05. ns = not statistically significant.

    Article Snippet: The following chemicals were used: doxycycline hyclate (Sigma Aldrich, D9891), RO3306 (AdipoGen Life Sciences, AGCR13515M), (R)-roscovitine (AdipoGen Life Sciences, AG-CR1-0006-M005), nocodazole (Selleckchem, S2775), (+)-S-trityl-L-cysteine (STLC) (Alfa Aesar, L14384), proTAME (Boston Biochem, I-440), apcin (Enamine, T0506-3874), taxol (Sigma-Aldrich, 33069-62-4), thymidine (Sigma-Aldrich, T9250), palbociclib (LC Laboratories, P-7722), torin1 (Cell Signaling, 14379S), rapamycin (RPI, R64500-0.001), MG-132 (Calbiochem, 474790), MG-262 (Apexbio, A8179-1), cycloheximide (Sigma, C7698), geranylgeranyl alcohol (Cayman Chemical, 13272), farnesyl alcohol (Cayman Chemical, 13268), puromycin (Thermo Scientific, 22742-0100), bafilomycin A1 (RPI, B40500-0.001), PFI-7 (Gift from C. H. Arrowsmith, Structural Genomics Consortium), SiR-DNA (SiR-Hoechst*) (Spirochrome, SC007), Pierce protease inhibitor tablet (Thermo Scientific, A32953), phosphatase inhibitor tablet (Thermo Scientific, A32957).

    Techniques: Ubiquitin Proteomics, Incubation