Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmid coding for E1 Ubiquitin-Activating Enzyme 1 (UBA1) (pCMV3-UBA1-c-Myc) was purchased from SiNo Biological (Düsseldorfer, Eschborn, Germany).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Cotransfection

Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Nucleic Acids Research

Article Title: CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

doi: 10.1093/nar/gkv434

Figure Lengend Snippet: DSB induced degradation of MDC1 happens more promptly in S-phase. ( A ) HEK 293T cells were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (5 Gy) and harvested at indicated time points. Left panel: the level of endogenous MDC1 was examined with indicated antibodies by immunoblotting. Blots were quantified using ImageJ (National Institutes of Health). Right panel: the cell cycle profiles of the cells are analyzed by flow cytometry. ( B ) U2OS cells were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (2 Gy) and probed with indicated antibodies at the indicated time point. Representative images are shown in the left panels, the quantification of the percentage of cells displaying MDC1 (upper) or Rad51 (lower) foci are shown in right panels. Error bars represent SEM. from three independent experiments. ** P < 0.01 two‐tailed Student's t ‐test. >200 cells were counted per experiment. ( C ) HEK 293T cells transfected with HA-MDC1 and FLAG-SUMO1 were synchronized in G1 phase by double thymidine block or released into S phase. Then the cells were treated with or without irradiation (5 Gy) and harvested at indicated time points. MDC1 was then immunoprecipitated with anti-HA antibody and MDC1 sumoylation was examined using anti-FLAG antibody. Blots were quantified using ImageJ (National Institutes of Health). ( D ) HEK 293T cells transfected with Myc-RNF4 were synchronized by double thymidine block or released into S phase. The cells were irradiated (5 Gy) and then collected at indicated time points. RNF4 was then immunoprecipitated and subjected to in vitro ubiquitination reactions in the presence of HA-Ub, UbcH5a and UBE1. The ubiquitination of RNF4 were analyzed by immunoblot as indicated.

Article Snippet: Sumoylated GST-MDC1(aa1818–2089) bound to GST-sepharose were washed with Tris-HCl (50 mM, pH7.5) and then incubated with 200 ng of UbcH5a (Boston Biochem), 300 ng of ubiquitin activating enzyme (UBE1) (Boston Biochem), 2 μg of HA-ubiquitin (Boston Biochem), 3 μg Myc-RNF4 or FLAG-RNF4 in 40 μl of reaction buffer (50 mM Tris[pH7.5], 2.5 mM MgCl 2 , 2 mM ATP, 2 mM DTT).

Techniques: Blocking Assay, Irradiation, Western Blot, Flow Cytometry, Two Tailed Test, Transfection, Immunoprecipitation, In Vitro, Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase

doi: 10.1093/nar/gkv434

Figure Lengend Snippet: The phosphorylation on RNF4 affects its activity. ( A ) Upper panel: HEK 293T cells transfected with FLAG-RNF4 WT, 2TA or 2TE mutant were synchronized in G1 phase by double thymidine block or released into S phase. The cells were irradiated (5 Gy) and then collected at indicated time points. RNF4 was then immunoprecipitated and subjected to in vitro ubiquitination reactions in the presence of HA-Ub, UbcH5a and UBE1. The ubiquitination of RNF4 were analyzed by immunoblot as indicated. Lower panel: the cell cycle profiles of the cells were analyzed by flow cytometry. ( B ) Upper panel: schematic of the combined in vitro sumoylation and ubiquitination assay procedures. Lower panel: bacterially expressed GST‐MDC1 (aa 1818–2089) bound to GST‐sepharose were subjected to combined in vitro sumoylation and ubiquitination reactions as shown in the upper panel. The GST‐MDC1(aa 1818–2089) was subjected to in vitro sumoylation reactions in the presence of SUMO1 and then subjected to in vitro ubiquitination reactions in the presence or absence of HA‐Ub, RNF4‐WT, RNF4‐2TA, or RNF4-2TE mutant as indicated. The ubiquitination of GST‐sepharose‐bound protein were analyzed by immunoblot as indicated. ( C ) Cells depleted RNF4 with siRNA were cotransfected with HA-MDC1 and FLAG-RNF4-WT, 2TA or 2TE mutant. The cells were synchronized in G1 phase by double thymidine block or released into S phase and then irradiated (5 Gy). The ubiquitination of HA-MDC1 was then analyzed with indicated antibodies.

Article Snippet: Sumoylated GST-MDC1(aa1818–2089) bound to GST-sepharose were washed with Tris-HCl (50 mM, pH7.5) and then incubated with 200 ng of UbcH5a (Boston Biochem), 300 ng of ubiquitin activating enzyme (UBE1) (Boston Biochem), 2 μg of HA-ubiquitin (Boston Biochem), 3 μg Myc-RNF4 or FLAG-RNF4 in 40 μl of reaction buffer (50 mM Tris[pH7.5], 2.5 mM MgCl 2 , 2 mM ATP, 2 mM DTT).

Techniques: Phospho-proteomics, Activity Assay, Transfection, Mutagenesis, Blocking Assay, Irradiation, Immunoprecipitation, In Vitro, Ubiquitin Proteomics, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Clr4 SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation

doi: 10.1038/s41467-024-53417-9

Figure Lengend Snippet: a Chromatin immunoprecipitation and sequencing (ChIP-seq) reads for H3K9me2 (blue), H3K9me3 (green) and RNA Pol II (Rpb1, red) were mapped to centromere 3 ( cnt3 ) right arm in the indicated strains. Relative enrichments of ChIP-seq reads from each strain were divided by the total count in WT. Scales for dcr1Δ ubc4-1 and clr4Δ H3K9me2 and H3K9me3 were expanded to visualize low reads. A schematic diagram of chromosome centromere 3 right arm showing innermost repeat ( imr ), outermost repeats ( dg , dh ) and boundary region ( irc ) is shown. Vertical red lines and pink boxes represent tRNA and euchromatic genes respectively. b , d Western blot (WB) analyses of immunoprecipitated Flag-Clr4 (Flag-Clr4 IP) in the indicated strains. The ratio of ubiquitinated Clr4 (Clr4-Ub) compared to unmodified Clr4 is indicated below each lane. Bottom, WB analysis of Flag-Clr4 from whole cell extract (WCE). Molecular weight markers are shown and uncropped images are provided in Source Data. c Schematic diagram of domain structure of Clr4 protein and the position of lysine residues. Ovals show the position of all 36 lysines of Clr4, which are arranged in 6 sub-groups. The second sub-group (2–8 K) is highlighted and amino acid sequences are shown. 2–4 K sub-group includes K109-K114. Red dots represent putative mono-ubiquitination sites identified by in vitro ubiquitination and Mass Spec analyses. CD: chromodomain, PS: PostSET. e ChIP-seq reads of H3K9me2 (blue) and H3K9me3 (green) mapped to chromosome 3 centromere right arm in the indicated strains. f sRNA-seq reads mapped to centromere 3 right arm in indicated strains. g Left, schematic diagram showing the presumptive subunit arrangement in the complex of Ubc4-CLRC and its substrate Clr4 for mono-ubiquitination. Red circle (U), ubiquitin. Right, model for the binding of Clr4 and Swi6 to existing H3K9me3 and the activity of Clr4 for converting H3K9me2 to H3K9me3. Blue circle, H3K9me2 and green circle, H3K9me3. Created in BioRender. Kim, H. (2024) BioRender.com/r28w184.

Article Snippet: For in vitro ubiquitination, recombinant 6xHis-Clr4 protein or 3xFlag-Clr4 purified form S. pombe cells were mixed with human Ubiquitin-activating Enzyme (UBE1, R&D systems E-305), human UbcH5c/UBE2D3 (R&D systems E2-627), human Ubiquitin (R&D systems U-100H) or human HA-Ubiquitin (R&D systems U-110), Mg-ATP (R&D systems B-20) and 10x E3 Ligase Reaction Buffer (R&D systems B-71) with or without CLRC complex purified from S. pombe cells.

Techniques: Chromatin Immunoprecipitation, Sequencing, ChIP-sequencing, Western Blot, Immunoprecipitation, Molecular Weight, In Vitro, Mass Spectrometry, Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Clr4 SUV39H1 ubiquitination and non-coding RNA mediate transcriptional silencing of heterochromatin via Swi6 phase separation

doi: 10.1038/s41467-024-53417-9

Figure Lengend Snippet: a ChIP-seq reads of Flag-Clr4 and RNA Pol II (Rpb1) and sRNA-seq reads mapped to chromosome 3 centromere ( cnt3 ) right arm in indicated strains. N/A, not available. b ChIP-qPCR assay showing enrichment of Flag-Clr4 at centromeric dh repeat in the indicated strains. Data are presented as mean ± SD ( n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P = 0.0271 for ubc4-1 , P = 0.0081 for cul4-1 and P < 0.0001 for rik1Δ, raf1Δ and raf2Δ . c RNA immunoprecipitation (RNA IP) and qPCR of Flag-Clr4 to centromeric dg and act1 transcript in the indicated strains. Data are presented as mean ± SD ( n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P < 0.0001 for ubc4-1 and cul4-1 ( dg , left). ns, not significant ( act1 , right). d Binding assay of in vitro ubiquitinated His-Clr4 protein with nucleosomes with unmodified histone H3 (me0) or H3K9me3 histone (me3) anchored to streptavidin beads. Input and His-Clr4 proteins from pull-down were analyzed by WB analyses using antibodies as shown. U, ubiquitin. HA-Ub, HA-ubiquitin. Schematic diagram (left). Molecular weight markers (KDa) are shown on left side of panels (right) and uncropped images are provided in a Source Data file. e Schematic diagram for tethering of GBD-Clr4- ΔCD to 5xUAS-ade6 locus with free Clr4 protein. Primers for ChIP-qPCR are indicated (left). Right, assay for silencing of ade6 on Low Ade medium in the indicated strains. Silencing (red color) depends on Ubc4 and Cul4. Bottom, ChIP-qPCR assays showing enrichment of H3K9me2 and H3K9me3 at ade6 in the indicated strains. Data are presented as mean ± SD (n = 3). P values are from one-way ANOVA test (Dunnett’s multiple comparisons test) without any adjustment. P < 0.0001 for 2, 3 and 4 (H3K9me2, Left). P < 0.0001 for 2, 3 and 4 (H3K9me3, Right). Created in BioRender. Kim, H. (2024) BioRender.com/r28w184.

Article Snippet: For in vitro ubiquitination, recombinant 6xHis-Clr4 protein or 3xFlag-Clr4 purified form S. pombe cells were mixed with human Ubiquitin-activating Enzyme (UBE1, R&D systems E-305), human UbcH5c/UBE2D3 (R&D systems E2-627), human Ubiquitin (R&D systems U-100H) or human HA-Ubiquitin (R&D systems U-110), Mg-ATP (R&D systems B-20) and 10x E3 Ligase Reaction Buffer (R&D systems B-71) with or without CLRC complex purified from S. pombe cells.

Techniques: ChIP-sequencing, RNA Immunoprecipitation, Binding Assay, In Vitro, Molecular Weight

Journal: Nature communications

Article Title: The Central Role of EED in the Orchestration of Polycomb Group Complexes

doi: 10.1038/ncomms4127

Figure Lengend Snippet: a, PRC1 proteins disassociate EED:EZH2 complexes. EZH2 protein was incubated with halo-tagged EED in the presence of different amounts of BMI1, RING1B or GUS proteins. Halo-Link PD followed by immunoblot analysis was performed. b , BMI1 and RING1B inhibit PRC2 mediated methylation of histone H3 in vitro . Indicated amounts of purified PRC2, RING1B, BMI1, and GUS were incubated with recombinant histone H3 with unmethylated H3K27 for two hours. Immunoblot analyses with anti-H3K27me3 and anti-H3K27me2 antibodies were then performed. c , BMI1 and RING1B inhibit PRC2 mediated methylation of nucleosomes in vitro . Indicated amounts of purified recombinant PRC2, RING1B, BMI1, and GUS proteins were incubated with nucleosomes containing unmethylated H3K27. ELISA with anti-H3K27me3 antibody was then performed. Assays were repeated thrice in triplicates. All error bars represent ±s.e.m. d , EED enhances RING1B mediated ubiquitination of histone H2A in vitro . Indicated amounts of purified EED, RING1B, BMI1, and GUS were incubated with recombinant histone H2A for one hour. Immunoblot analyses with anti-uH2A antibody were then performed to measure histone H2A ubiquitin E3 ligase activity. Assays were independently repeated three times. Normalized band intensity by densitometry from the three independent assays is represented as bar graphs below the immunoblot. Error bars represent ±s.e.m. e, EED enhances PRC1 mediated ubiquitination of nucleosome H2A in vitro . Purified EED, RING1B, BMI1, and GUS were incubated with reconstituted mono-nucleosomes with un- or tri-methylated H3K27. Immunoblot analyses with anti-uH2A antibody were then performed to measure histone H2A ubiquitin E3 ligase activity. Normalized band intensity by densitometry from three independent assays is represented as bar graphs blow the immunoblot. Error bars represent ±s.e.m. f , Knockdown of EED and RING1B altered H3K27me3 and uH2A levels in cells. EED- and RING1B-specific siRNA duplexes and non-targeting control were transfected into DU145 cells. Expression levels of indicated proteins were assessed by immunoblot analysis. Normalized band intensity by densitometry from three independent assays is represented as bar graphs blow the immunoblot. All error bars represent ±s.e.m.

Article Snippet: Recombinant Ubiquitin Activating Enzyme (UBE1), UbcH5c Conjugating Enzyme E2, Ubiquitin, and Ubiquitin Conjugation Reaction Buffer Kit were purchased from Boston Biochem, Inc. (Cambridge, MA).

Techniques: Incubation, Western Blot, Methylation, In Vitro, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Ubiquitin Proteomics, Activity Assay, Knockdown, Control, Transfection, Expressing

Journal: Nature communications

Article Title: The Central Role of EED in the Orchestration of Polycomb Group Complexes

doi: 10.1038/ncomms4127

Figure Lengend Snippet: EED is critical for PRC2 to methylate histone H3 at lysine 27. Once H3K27 are methylated, EED directly recruits PRC1 to the tri-methylated H3K27 loci and enhances PRC1 ubiquitin E3 ligase activity.

Article Snippet: Recombinant Ubiquitin Activating Enzyme (UBE1), UbcH5c Conjugating Enzyme E2, Ubiquitin, and Ubiquitin Conjugation Reaction Buffer Kit were purchased from Boston Biochem, Inc. (Cambridge, MA).

Techniques: Methylation, Ubiquitin Proteomics, Activity Assay