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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.
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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