Journal: Molecular Cell

Article Title: Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

doi: 10.1016/j.molcel.2018.09.028

Figure Lengend Snippet: The RING Finger and UBL Domain of UHRF1 Are Required for Stabilizing the E2/E3/Chromatin Complex (A) GST-UbcH5a pull-down assays indicate that UHRF1 alone shows very little binding to UbcH5a. Binding of UHRF1 to GST-UbcH5a is stimulated in the presence of 12 × 187 bp chromatin arrays but less so by mono-nucleosomes. Similar results are seen when comparing UHRF1 WT and UHRF1 ΔMW . (B) GST-pull-down assays using UHRF1 (ΔMW and the indicated mutants) in the absence or presence of 12 × 187 bp chromatin arrays. Binding was analyzed as in (A). (C) The binding of UHRF1 variants in the presence of 12 × 187 bp arrays relative to GST-UbcH5a, shown in (B), was quantified using ImageJ in at least 3 independent experiments and normalized to the UHRF1 ΔMW protein. The mean was plotted ± the SEM.

Article Snippet: Wild-type human UbcH5a (UBE2D1) was synthesized by Genscript and cloned into a pRSFDuet1 vector with a Prescission protease cleavable N-terminal His 6 -tag.

Techniques: Binding Assay

Journal: Molecular Cell

Article Title: Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

doi: 10.1016/j.molcel.2018.09.028

Figure Lengend Snippet: XL-MS Experiments to Probe the Conformational Changes in UHRF1 upon Chromatin and E2 Binding (A–E) XL-MS diagrams of UHRF1 in isolation (A), with mono-nucleosomes (B), 12 × 187 bp chromatin arrays (C), UbcH5a/UBE2D1 (D), and in the context of 12 × 187 bp arrays and UbcH5a (E). Intra-protein crosslinks are in red and inter-protein crosslinks in blue (histones) or green (UbcH5a). XL-MS experiments with UHRF1 WT are on the left and UHRF1 ΔMW on the right. Schematic representations of the proposed UHRF1 conformation in each experiment are shown with arrows indicating the predominant crosslinks from UHRF1 to histones and UbcH5a. UHRF1 domains are color coded as in Figure 1 A, with histones in yellow and UbcH5a in green. has the list of crosslinks identified in each experiment, and has an example spectrum as output from StavRox.

Article Snippet: Wild-type human UbcH5a (UBE2D1) was synthesized by Genscript and cloned into a pRSFDuet1 vector with a Prescission protease cleavable N-terminal His 6 -tag.

Techniques: Structural Proteomics, Binding Assay, Isolation

Journal: Molecular Cell

Article Title: Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

doi: 10.1016/j.molcel.2018.09.028

Figure Lengend Snippet: The E3 Activity of UHRF1 Is Dependent on a Hydrophobic Patch on the UBL and a Regulatory Ubiquitin Binding Surface on the E2 (A) (Top panel) Atomic space-fill structures of ubiquitin (PDB: 1UBQ ) and the UHRF1 UBL domain (PDB: 2FAZ ) color coded by hydrophobicity. Orange-red represents more hydrophobic residues, whereas blue residues are more polar. (Bottom panel) Sequence alignment of human ubiquitin with the UBL domain of UHRF1 from various species and the UBL domain of human Parkin. F46 in the UHRF1 UBL and corresponding isoleucines in ubiquitin and Parkin are indicated in red. (B) E3 ubiquitin ligase assays carried out with the indicated mutants over 1 hr at 25°C. H3 ubiquitylation is drastically reduced in the F46A mutant with some UHRF1 auto-ubiquitylation observed. The S22R mutation in UbcH5a abrogates ubiquitylation of the substrate (UHRF1 or histone H3) in the context of both UHRF1 WT and UHRF1 ΔMW . (C) GST-UbcH5a pull-downs using UHRF1 WT and the F46A mutant. Binding assays were carried out as described in Figure 4 C. Quantification of three independent experiments indicates reduced binding of F46A UHRF1 to UbcH5a relative to the wild-type in the presence of 12 × 187 bp arrays. The mean was plotted ± the SEM. (D) GST-UbcH5a pull-downs using UHRF1 WT with wild-type UbcH5a and the S22R mutant in the presence of 12 × 187 bp arrays. Quantification of three independent experiments reveals reduced binding of UHRF1 to S22R UbcH5a compared with the wild-type. The mean was plotted ± the SEM. (E) Proposed model for the contact between the hydrophobic patch on the UBL domain of UHRF1 (indicated in orange) and the regulatory region on UbcH5a centered on Ser-22.

Article Snippet: Wild-type human UbcH5a (UBE2D1) was synthesized by Genscript and cloned into a pRSFDuet1 vector with a Prescission protease cleavable N-terminal His 6 -tag.

Techniques: Activity Assay, Ubiquitin Proteomics, Binding Assay, Sequencing, Mutagenesis

Journal: Molecular Cell

Article Title: Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

doi: 10.1016/j.molcel.2018.09.028

Figure Lengend Snippet: A Hydrophobic Patch Mutant of the UBL Domain Does Not Support UHRF1-Dependent DNA Methylation in Mouse ESCs (A) DNMT1 localization during DNA replication assessed by immunofluorescence in Uhrf1 WT , Uhrf1 −/− , and Uhrf1 mutant ( Uhrf1 H730A /clone 2C11 and Uhrf1 F46A /clone 1D7) mESCs. Newly synthesized DNA in S phase cells was visualized with fluorescently labeled EdU. Scale bars for individual nuclei and pool of cells are 5 μm and 10 μm, respectively. (B) Western blot analysis (top panel) of the expression of UHRF1 and DNMT1 in the mESCs used to measure the effect of UBL and RING domain point mutants on DNA CpG methylation (lower panel) and DNMT1 localization to nuclear replication foci shown in (A). DNA methylation levels at LINE-1 elements (%) as measured by targeted bisulfite sequencing (TaBA-seq) of n = 4 biological replicates of wild-type J1 ( Uhrf1 WT ), Uhrf1 −/− , Uhrf1 F46A , and Uhrf1 H730A mESCs are shown. Dashed red line indicates the median methylation level in WT J1 ESCs. Boxes indicate the 25th to the 75th percentile and whiskers represent 1.5-fold interquartile range (IQR). Outliers are indicated by dots above and below the whiskers. Welch two-sided t test: ∗∗∗ p < 0.001. (C) Co-immunoprecipitation experiments of C-terminally CFP-tagged UHRF1 and the indicated mutants with the mCherry-tagged RFTS domain of DNMT1. Constructs were co-transfected and immunoprecipitated from 293T cells. (D) Proposed model of the mechanism of UHRF1-mediated H3 ubiquitylation on chromatin. UHRF1 engages chromatin via an interaction of the TTD-PHD module with histone H3 methylated at Lys-9 (blue square) and SRA-mediated binding to a hemi-methylated CpG in the linker DNA (open circle). UbcH5a can bind to UHRF1 in this configuration with the UBL hydrophobic patch (orange) contacting the “backside” of UbcH5a. This complex is stabilized by neighboring nucleosomes. This positions the E2∼Ub for efficient transfer of ubiquitin to H3 (Lys-18 and Lys-23). DNMT1 binds the ubiquitylated H3 via ubiquitin-interacting motifs in its RFTS domain (purple) to maintain DNA CpG methylation.

Article Snippet: Wild-type human UbcH5a (UBE2D1) was synthesized by Genscript and cloned into a pRSFDuet1 vector with a Prescission protease cleavable N-terminal His 6 -tag.

Techniques: Mutagenesis, DNA Methylation Assay, Immunofluorescence, Synthesized, Labeling, Western Blot, Expressing, CpG Methylation Assay, Methylation Sequencing, Methylation, Immunoprecipitation, Construct, Transfection, Binding Assay, Ubiquitin Proteomics

Journal: Molecular Cell

Article Title: Critical Role of the UBL Domain in Stimulating the E3 Ubiquitin Ligase Activity of UHRF1 toward Chromatin

doi: 10.1016/j.molcel.2018.09.028

Figure Lengend Snippet:

Article Snippet: Wild-type human UbcH5a (UBE2D1) was synthesized by Genscript and cloned into a pRSFDuet1 vector with a Prescission protease cleavable N-terminal His 6 -tag.

Techniques: Recombinant, Ubiquitin Proteomics, Methylation, Picogreen Assay, Western Blot, Immunofluorescence, CpG Methylation Assay, Clone Assay, Modification, Mutagenesis, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Specific genomic targeting of the RNF12/RLIM E3 ubiquitin ligase selectively programmes developmental transcription

doi: 10.1101/2023.06.14.544957

Figure Lengend Snippet: (A) Schematic representation of mouse RNF12 BR, BR1, BR2 and BR3 deletions. (B) Immunoblotting for HA-tagged mRNF12 on soluble and chromatin fraction of transfected RNF12 knock-out ( Rlim -/y ) mESCs with HA-tagged versions of RNF12 WT and RNF12 BR deletions. Data are representative of n=5 independent experiments. (C) Graph representing the quantification of the HA-mRNF12 signal observed in the chromatin fraction in B. The percentage of each deletion mutant in the chromatin fraction was normalized to that observed for RNF12 WT. Data represented as mean ± SEM (n=5). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (D) In vitro REX1 substrate ubiquitylation assay of RNF12 WT and RNF12 BR deletions. Top: Fluorescently labelled ubiquitylated proteins were detected by scan at 680nm (Cy5-Ub) after 30 mins of reaction, and specific ubiquitylated REX1 (REX1-Ub n ) and RNF12 (RNF12-Ub n ) signals are indicated. Bottom: Immunoblot analysis of RNF12 (using anti-RNF12 mouse monoclonal antibody) and REX1 protein levels. Data are representative of n=4 independent experiments. (E) REX1 ubiquitylation signal was quantified and normalized to total REX1 amounts. Only the first three ubiquitylated bands (REX1-Ub 3 ) were quantified. Data represented as mean ± SEM (n=4). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (F) Immunoblotting for HA and FLAG in HA immunoprecipitates from RNF12 and REX1 double knock-out ( Rlim -/y ; Zfp42 -/- ) mESCs expressing FLAG-REX1 with either empty vector (control) or the indicated HA-RNF12 deletions mutants. RNF12 and REX1 double knock-out ( Rlim -/y ; Zfp42 -/- ) mESCs were treated with MG132 inhibitor for 2 h and Ponceau S staining is shown as a loading control. Data are representative of n=4 independent experiments. (G) RNF12 knock-out ( Rlim -/y ) mESCs expressing HA-tagged mouse RNF12 WT or BR deletions were treated with either DMSO (vehicle control) or MG132 for 1 h. REX1 ubiquitylation levels are shown. Data are representative of n=4 independent experiments. (H) REX1 ubiquitylation signal on the chromatin fraction in the presence of MG132 in G was quantified and normalized to the signal observed after transfection of HA-mRNF12 WT in RNF12 knock-out ( Rlim -/y ) mESCs. Data represented as mean ± SEM (n=4). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (I) HA-mouse RNF12 and REX1 half-life was analyzed by immunoblotting in RNF12 knock-out ( Rlim -/y ) mESCs expressing HA-tagged mouse RNF12 WT, RNF12 ΔBR, RNF12 ΔBR1, RNF12 ΔBR2 and RNF12 ΔBR3. Cells were treated with 350 µM cycloheximide (CHX) for the indicated times. Ponceau S staining is shown as a loading control. Data are representative of n=4 independent experiments. (J) Representation of the HA-tagged RNF12 and REX1 proteins levels quantified in I. Protein levels were normalized to the Ponceau S loading control. Data represented as mean ± SEM (n=4). Statistical significance of each deletion mutant compared to HA-mRNF12 WT was determined at 2 h for HA-tagged mouse RNF12 and at 4 h for REX1 by paired t test; two-sided, confidence level 95%. HA-tagged mRNF12: RNF12 ΔBR (**) p=0.0026, RNF12 ΔBR1 (ns) p=0.6554, RNF12 ΔBR2 (*) p=0.0393 and RNF12 ΔBR3 (*) p=0.0186. REX1: RNF12 ΔBR (*) p=0.0322, RNF12 ΔBR1 (ns) p=0.7590, RNF12 ΔBR2 (**) p=0.0050 and RNF12 ΔBR3 (ns) p=0.2781.

Article Snippet: RNF12 recombinant protein (140 nM) was incubated with 20 µl ubiquitylation mix containing 0.1 µM UBE1, 0.05 µM UBE2D1 (UBCH5A), 1.5 µg REX1, 2 µM Cy5-Ubiquitin (South Bay Bio), 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP) pH 7.5, 5 mM ATP, 50 mM Tris pH 7.5 and 5 mM MgCl 2 .

Techniques: Western Blot, Transfection, Knock-Out, Mutagenesis, In Vitro, Ubiquitin Assay, Expressing, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Specific genomic targeting of the RNF12/RLIM E3 ubiquitin ligase selectively programmes developmental transcription

doi: 10.1101/2023.06.14.544957

Figure Lengend Snippet: (A) Schematic representation of mouse RNF12 N-terminal deletion (ΔN), and RNF12 N1, N2 and N3 deletions. (B) Immunoblotting for HA-tagged mouse RNF12 in soluble and chromatin fractions of RNF12 knock-out ( Rlim -/y ) mESCs expressing HA-tagged mouse RNF12 WT and N-terminal deletions. Data are representative of n=5 independent experiments. (C) Graph representing the quantification of the HA-RNF12 signal observed in the chromatin fraction. The percentage of each deletion mutant in the chromatin fraction was normalised to that observed for mRNF12 WT. Data represented as mean ± SEM (n=5). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (D) RNF12 knock-out ( Rlim -/y ) mESCs expressing HA-tagged mouse RNF12 WT and N-terminal deletions were treated with MG132 for 1 h. REX1 ubiquitylation levels are shown. Data are representative of n=4 independent experiments. (E) REX1 ubiquitylation signal in the chromatin fraction in the presence of MG132 was quantified and normalized to the signal observed after transfection of HA-RNF12 WT in Rlim -/y mESCs. Data represented as mean ± SEM (n=4). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (F) In vitro REX1 substrate ubiquitylation assay of WT mRNF12 and mRNF12 N-terminal deletion at different amounts. Top: Fluorescently labelled ubiquitylated proteins were detected by scan at 680nm (Cy5-Ub) after 30 mins of reaction and specific ubiquitylated REX1 (REX1-Ub n ) and RNF12 (RNF12-Ub n ) signals are indicated. Bottom: Immunoblot analysis of RNF12 (using anti-RNF12 mouse monoclonal antibody) and REX1 protein levels. Data are representative of n=5 independent experiments. (G) REX1 ubiquitylation signal was quantified and normalized to total REX1 amounts. Only the first three ubiquitylated bands (REX1-Ub 3 ) were quantified. Data represented as mean ± SEM (n=4). Statistical significance was determined by paired t test; two-sided, confidence level 95%. (H) REX1 half-life was analyzed by immunoblotting of RNF12 knock-out ( Rlim -/y ) mESCs expressing either empty vector (control), HA-mRNF12 WT and HA-mRNF12 N-terminal deletion. Cells were treated with 350 µM cycloheximide (CHX) for the indicated times. Ponceau staining is shown as a control. Data are representative of n=5 independent experiments. (I) Representation of the REX1 proteins levels quantified in H. Protein levels were normalized to the Ponceau control. Data represented as mean ± SEM (n=5). Statistical significance of REX1 stability in Rlim -/y mESCs expressing HA-mRNF12 N-terminal deletion compared to those expressing HA-mRNF12 WT was determined at 2 h by paired t test; two-sided, confidence level 95%.

Article Snippet: RNF12 recombinant protein (140 nM) was incubated with 20 µl ubiquitylation mix containing 0.1 µM UBE1, 0.05 µM UBE2D1 (UBCH5A), 1.5 µg REX1, 2 µM Cy5-Ubiquitin (South Bay Bio), 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP) pH 7.5, 5 mM ATP, 50 mM Tris pH 7.5 and 5 mM MgCl 2 .

Techniques: Western Blot, Knock-Out, Expressing, Mutagenesis, Transfection, In Vitro, Ubiquitin Assay, Plasmid Preparation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: ACR and the AL2CR bind an active CRL4CRBN. A, St, St-ACR, St-AL1CR, and St-AL2CR mouse brain pulldowns were incubated with (+) or without (−) ubiquitin-FLAG (Ub-FLAG), E2, and/or E1. After the reaction, these in vitro ubiquitination assays were probed on WB with an anti-FLAG antibody. E2 promotes ubiquitination in St-ACR and St-AL2CR pulldowns. The WB shown is representative of five independent experiments. B, WB with an α-CRBN antibody indicates the in vitro ubiquitination assays of St-ACR brain pulldowns where Crbn is poly-ubiquitinated, indicating that St-ACR isolates from brain an active CRL4CRBN. The WB shown is representative of two independent experiments.

Article Snippet: Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Incubation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: In vitro ubiquitination of proteins present in the St-ACR Tyr(P)Thr(P) pulldowns 1st column lists some of the proteins ubiquitinated in vitro in an ACR Tyr(P)Thr(P) -dependent manner. 2nd column lists all the lysine residues found ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in previous UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported online. 3rd column reports database accession numbers.

Article Snippet: Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: APP could be both a substrate and a substrate recognition subunit of Stub1 and CRL4CRBN E3 protein ligases. A, CRL4CRBN mediates ubiquitination of lysine residue(s) present in the cytoplasmic tail of APP (with Lys756 being the most likely candidate). B, Stub1 could also be involved in the ubiquitination of cytoplasmic APP lysine residue(s). C, APP could bridge cytosolic and membrane-bound proteins to CRL4CRBN de facto functioning as a substrate recognition unit of a CRL4CRBN-APP E3 ubiquitin-protein ligase. D, in this final model APP is postulated to act as a substrate recognition unit for a Stub1-APP E3 ubiquitin-protein ligase, mediating ubiquitination of cytosolic and/or membrane-bound proteins that interact with the ACR ubiquitination of APP, and APP-binding proteins could lead to functional modification and/or to degradation of the ubiquitinated proteins by the proteasome, autophagosomes, and/or lysosomes. E, some of the APP-derived metabolites that contain the ACR and can potentially interact with Stub1 and CRL4CRBN. Processing of full-length APP by β-, α-, and γ-secretase can have several functional consequences. For example, it is possible that full-length APP-Stub1/CRL4CRBN, β-CTF-Stub1/CRL4CRBN, and α-CTF-Stub1/CRL4CRBN complexes have distinct functions, i.e. that the ectodomain of APP may influence the function of the ACR. That processing of β-CTF and α-CTF by γ-secretase could have functional consequences is obvious. Indeed, AID-Stub1/CRL4CRBN complexes are released from membranes. This may, among other things, result into down-modulation of the APP-dependent ubiquitination of trans-membrane proteins. F, cleavage of APP-Stub1/CRL4CRBN, β-CTF-Stub1/CRL4CRBN, α-CTF-Stub1/CRL4CRBN, and AID-Stub1/CRL4CRBN by caspases could functionally separate the activities linked to the various ACR-Stub1 and ACR-CRL4CRBN complexes.

Article Snippet: Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Binding Assay, Functional Assay, Modification, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, Cul4a, and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Ddb1, Crbn, Cul4a, and Cul4b bind to Ccas and Stub1 binds to JCasp Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of Ddb1, Crbn, Cul4a, and Cul4b, whereas binding of Pin1 and Grb2 is dependent on phosphorylation of Thr 668 and Tyr 682 , respectively. Several of the other UPS-related proteins that are bound to the ACR bind to either Ccas or JCasp or both.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: In vitro ubiquitination of proteins present in the St-ACR Tyr(P)Thr(P) pulldowns 1st column lists some of the proteins ubiquitinated in vitro in an ACR Tyr(P)Thr(P) -dependent manner. 2nd column lists all the lysine residues found ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in previous UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported online. 3rd column reports database accession numbers.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: In Vitro

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A Representative statistics analysis of CIB1 levels in two different data using the ONCOMINE database ( www.oncomine.org ). Data were filtered to display upregulation of CIB1 in LAC tissues compared to normal tissues (T: Tumor, N: normal tissues). a: Hou Lung Statistics; b: Landi Lung Statistics. B Representative immunohistochemically stained images of normal and LAC tissue using anti-CIB1 antibody: a, b: negative expression of CIB1 in normal lung tissue; c, d: negative expression of CIB1 in lung adenocarcinoma; e, f: weakly positive expression of CIB1 in lung adenocarcinoma; g, h: moderately positive expression of CIB1 in lung adenocarcinoma; i, j: Strong positive expression of CIB1 in lung adenocarcinoma;. C Relative mRNA expression level of CIB1 in LAC tumor tissue compare to normal tissues from 60 patients. D Quantitative RT-PCR results showed CIB1 expression levels in five LAC cell lines and normal bronchus cell lines. E Western blots results showed CIB1 expression levels in five LAC cell lines and normal bronchus cell lines. F , G Metastatic or advanced TNM stage LAC tumors had higher CIB1 expression levels compared with those of nonmetastatic or early TNM stage LAC tumors. H , I Kaplan–Meier analysis showed that the 5-year overall survival and disease-free survival for LAC patients with higher CIB1 expression levels were significantly shorter than those of patients with lower CIB1 expression level. * P < 0.05; ** P < 0.01; *** P < 0.001; HR, hazard ratio.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: Correlation analysis between CIB1 expression and clinical characteristics.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Expressing

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: Cox regression analysis of overall survival (OS) and disease-free survival (DFS) in 60 patients with LAC cancer.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Adjuvant, Expressing

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A , B Representative images and quantification of the Transwell assay using CIB1-upregulation A549/H1299 cells. C , D Representative images and quantification of the Transwell using anti-CIB1-transfected H1437/PC-9 cells. E Quantification of wound healing assays using CIB1/anti-CIB1-transfected LAC cells. F Western blot assay showing AKT (pan), p-AKT, E- cadherin, N-cadherin, and Vimentin protein level in CIB1/anti-CIB1-transfected LAC cells. Cells were treated with 5 μM MK2206 for 24 h. * P < 0.05 vs negative control (NC) group, ** P < 0.01 vs NC group, *** P < 0.001 vs NC group.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transwell Assay, Transfection, Western Blot, Negative Control

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: CIB1 and CHIP specifically interact. A SDS-PAGE gel of proteins bound to IgG (left lane) or CIB1 (right lane). Protein marker stands stand for (from top to bottom): 170, 130, 100, 70 (Red), 55, 40, 35, 25, 15/10 kDa. B Endogenous interaction between CIB1 and CHIP in PC-9 cells. C PC-9 cells were transiently transfected for 36 h with CHIP plasmid, and cell lysates were immunoblotted with indicated antibodies. D HEK293 cells were transiently transfected for 36 h with Flag-CIB1 and Myc-CHIP, and cell lysates were immunoblotted with indicated antibodies. E Representative immunohistochemically stained images of LAC tissues using the anti-CHIP and anti-CIB1 antibodies. Areas in the black squares are magnified in the right slide panels. F Pearson analysis correlation between CIB1 and CHIP expression in LAC tissues ( r = −0.3582, P = 0.0049) G HEK293 cells were transfected with Flag-CIB1 (1 μg) and various concentrations of Myc-CHIP plasmids (0, 0.2, 0.5, and 1 μg). And after 36 h, cell lysates were immunoblotted with anti-Flag, anti-Myc, and anti-Tublin antibodies. H Representative confocal images of immunostaining for CHIP (green) and CIB1 (red) in PC-9 and A549 cells. Scale bar, 50 μm. I Cells were treated with 40 μg/ml cycloheximide for the indicated times, and cell lysates were immunoblotted with indicated antibody. J HEK293 cells were transfected for 36 h with Flag-CIB1 alone or together with Myc-CHIP and treated with MG-132 (10 μM), or leupeptin (100 μM) for 4 h. Cell lysates were analyzed by western blotting with the indicated antibodies.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: SDS Page, Marker, Transfection, Plasmid Preparation, Staining, Expressing, Immunostaining, Western Blot

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A PC-9 were transfected with CHIP plasmid for 36 h. Cell extracts were immunoprecipitated with the anti-CIB1 antibody, followed by immunoblotting with the anti-Ubiquitin antibody. B HEK293 cells were transfected with HA-ubiquitin, Flag-CIB1 with or without Myc-CHIP. Cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with anti-flag antibodies. C PC-9 cells were transfected with CHIP or CHIP-ΔUbox plasmid. Cell extracts were immunoprecipitated with the anti-ubiquitin antibody, followed by immunoblotting with the anti-CIB1 antibody. D HEK293 cell were transfected with Flag-CIB1, HA-ubiquitin, with or without Myc-CHIP or Myc -CHIP-ΔUbox. Denatured cell lysates were immunoprecipitated with the anti-HA antibody, followed by immunoblotting with Flag antibodies. E Where specified, purified E1, E2, ubiquitin, CHIP and CIB1 proteins were incubated with in vitro ubiquitination buffers. Reaction samples were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Sliver staining or immunoblotting with the indicated antibodies. F HEK293 cells were transfected for 36 h with Flag-CIB1, Myc-CHIP, HA-Ubiquitin or HA-K48R-ubiquitin or HA-K63R-ubiquitin. Cell extracts were immunoprecipitated with the anti-HA antibody followed by immunoblotting with the anti-Flag antibody.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Purification, Incubation, In Vitro, Polyacrylamide Gel Electrophoresis, Staining

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A HEK293 cells were co-transfected with HA-ubiquitin and Flag-CIB1 and its corresponding mutated plasmids for 36 h. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. B HEK293 cells were co-transfected with HA-ubiquitin with HA-ubiquitin, and Flag-CIB1 or Flag-K10R-CIB1 or Flag-K65R-CIB1 or Flag-K10R+K65R-CIB1. Cell lysates were subjected to immunoprecipitation with HA antibody, followed by Flag immunoblotting. C HEK293 cells were co-transfected with Flag-CIB1, Flag-K10R-CIB1, Flag-K65R-CIB1, Flag-K10R+K65R-CIB1 and Myc-CHIP. Cell lysates were analyzed by immunoblotting with MYC, Flag and α-Tublin antibodies. D Computational modeling results show multiple binding sites for ubiquitin on CIB1. The red region is the binding site for CIB1.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Binding Assay

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A , B Representative images and quantification of the Transwell assays using transfected A549/H1299 cells. C , D Quantification of the wound healing assays using transfected A549/H1299 cells. E Western blot assay showing the phosphorylation of AKT and EMT enhanced by CIB1. Restoration of CHIP attenuated this elevation in LAC cells.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Transfection, Western Blot, Phospho-proteomics

Journal: Cell Death and Differentiation

Article Title: CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal transition and tumor metastasis in lung adenocarcinoma

doi: 10.1038/s41418-020-00635-5

Figure Lengend Snippet: A Representative luciferase images and quantification of average luciferase intensity of lungs in the i.v. metastasis assay. B , C Representative photographs and quantification of metastatic tumor nodes in mouse lungs from the i.v. metastasis assay. D Representative immunohistochemically stained images of lung tissue using anti-CIB1, anti-E-cadherin, and N-cadherin antibody. E CHIP-mediated CIB1 ubiquitination regulated epithelial–mesenchymal and tumor metastasis in lung adenocarcinoma through AKT pathway. * P < 0.05 vs negative control (NC) group, ** P < 0.01 vs NC group.

Article Snippet: 2.5 μl 10× E3 Ligase Reaction Buffer (B-71, Boston Biochem), 1 μl Recombinant Human Ubiquitin Protein (U-100H-10M, Boston Biochem), 10 mM MgATP Solution (B-20, Boston Biochem), 100 nM Recombinant Human His6-Ubiquitin E1 Enzyme (E-304–05, Boston Biochem), 1 μM Recombinant Human UbcH5a/UBE2D1 Protein (E2–616–100, Boston Biochem), 1 μM Recombinant Human CHIP/STUB1 Protein (E3–220–050, Boston Biochem), 5 μM CIB1 Fusion Protein (Ag2391, Protein Tech) was added in a microcentrifuge tube and be incubated in a 37 °C water bath for 60 min. Then the lysis was added by SDS-PAGE sample buffer and followed by electrophoresis.

Techniques: Luciferase, Staining, Ubiquitin Proteomics, Negative Control