Journal: The Journal of Cell Biology

Article Title: Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

doi: 10.1083/jcb.201506048

Figure Lengend Snippet: TRIM3 polyubiquitylates ACTG1. (A–D) TPA stimulation induces ACTG1 polyubiquitylation by TRIM3 in HEK293 cells. HEK293 cells were transfected with TRIM3 or ΔRBCC-TRIM3. After incubation for 4 h in the presence of MG132, cells were lysed and ACTG1 was immunoprecipitated from the lysates, resolved on SDS-PAGE, and immunoblotted. Blots were first stained for polyubiquitin (A) and then stripped and restained for ACTG1 (B). Under these basal conditions, only unmodified ACTG1 was detected. However, when cells were incubated in the presence of MG132 and TPA, a significant increase in high-molecular-weight polyubiquitylated forms of ACTG1 (indicated with *) was observed specifically in TRIM3-transfected cells (C–E; means ± SEM, two-tailed t test, *, P < 0.05, n = 3 cultures per condition). (F) TPA induces ACTG1 polyubiquitylation by TRIM3 in hippocampal neurons. Cultured hippocampal neurons from wild-type and Trim3 −/− mice were treated with MG132 and TPA for 4 h. Lysates were immunoblotted and stained for ACTG1. TPA induced the appearance of multiple high molecular weight (50–100 kD) bands (indicated with *) consistent with polyubiquitylation and specifically in neurons from wild-type mice and not from Trim3 −/− mice. (G) Actg1 mRNA is present in TRIM3/PURA containing mRNP granules. mRNP granules were immunoprecipitated from hippocampal lysates with antibodies against TRIM3 or PURA. mRNA was isolated from immunoprecipitates, reverse transcribed into cDNA, and used for real-time quantitative PCR. Actg1 and Actb mRNA was detected at equal levels in all precipitates, whereas the negative control mRNA Slc1a3 was ∼10-fold lower detected (means ± SEM, n = 3 immunoprecipitations per condition).

Article Snippet: After 14 d in culture, neurons were either lysed in SDS loading buffer directly or first incubated with 20 μM MG132 (Tocris Bioscience) and 500 nM TPA (Sigma-Aldrich) for 4 h and then lysed.

Techniques: Transfection, Incubation, Immunoprecipitation, SDS Page, Staining, High Molecular Weight, Two Tailed Test, Cell Culture, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) Histogram showing the frequency of PPM1D amplifications and C-terminal truncating mutations by cancer tissue type acquired from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Red box indicates PPM1D copy number gain, and blue box indicates PPM1D truncating mutation. (B) Lollipop plot showing the distribution of PPM1D C-terminal truncating mutations obtained from the COSMIC database. Green dot indicates PPM1D frameshift mutation, and gray dot indicates PPM1D nonsense mutation. Mutations with numbers of reported above 20 were labeled. (C) Western blot of PPM1D in MOLM13 wildtype (wt), MOLM13 truncated mutant (sg PPM1D exon6 ), U2OS, or HCT116 cells. (D) Western blot (top) and quantification (bottom) of a cycloheximide (CHX) chase time-course of PPM1D in MOLM13-wt, MOLM13-sg PPM1D exon6 , U2OS, or HCT116. (E) Western blot of PPM1D, ubiquitin, and LC3B in MOLM13-wt, MOLM13-sg PPM1D exon6 , U2OS, or HCT116 cells after treatment with CHX, MG132, or Bafilomycin A1. (F) Western blot of PPM1D and ubiquitin in MOLM13-wt, MOLM13-sg PPM1D exon6 , U2OS, or HCT116 cells after treatment with CHX, MG132, or TAK-243. (G) Schematic of immunoprecipitation mass spectroscopy experiment where V5-tr-PPM1D or V5-fl-PPM1D were expressed in HEK293T cells (left) and quantification (right) of ubiquitinated lysines across replicates. (H) V5-fl-PPM1D or V5-tr-PPM1D were co-expressed with HA-Ubiquitin in HEK293T cells followed by native or denaturing immunoprecipitation of HA-Ubiquitin and immunoblotting for V5-PPM1D. Whole cell extract (WCE) is shown below. (I) Western blot (top) and quantification (bottom) of a CHX chase time-course of HA-PPM1D in HEK293T cells expressing HA-fl-PPM1D, HA-fl-PPM1D-K 0 , HA-tr-PPM1D, or HA-tr-PPM1D-K 0 .

Article Snippet: Cells were then collected and lysed in NP40 buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40) supplemented with 10 μM MG132, 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 78438) and 10 mM N-Ethylmaleimide (MedChemExpress, HY-D0843).

Techniques: Mutagenesis, Labeling, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Mass Spectrometry, Expressing

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) GFP:mCherry quantification by flow cytometry in MOLM13 cells expressing the EGFP-tr-PPM1D-IRES- mCherry or EGFP-fl-PPM1D-IRES-mCherry or EGFP-PPM1D 400-605 -IRES-mCherry stability reporter at baseline (left) or after treatment with MG132 (right). (B) Schematic of a genome-wide CRISPR screen in U937 cells expressing the EGFP-fl-PPM1D-IRES-mCherry or EGFP-PPM1D 400-605 -IRES-mCherry stability reporter treated with DMSO or cycloheximide (CHX). (C) Volcano plot of a genome-wide CRISPR screen in U937 cells expressing the EGFP-fl-PPM1D-IRES-mCherry stability reporter treated with DMSO or CHX. (D) Volcano plot of a genome-wide CRISPR screen in U937 cells expressing the EGFP-PPM1D 400-605 -IRES- mCherry stability reporter treated with DMSO or CHX. (E) Schematic of a BirA-based proximity ligation assay in which HEK293T cells expressing BirA-tr-PPM1D or BirA-fl-PPM1D were incubated with biotin, followed by MG132 treatment. Biotinylated substrates were enriched by streptavidin immunoprecipitation and identified by mass spectrometry. (F) Volcano plot of a BirA-based proximity ligation assay in HEK293T cells expressing BirA-tr-PPM1D or BirA-fl- PPM1D. (G) Western blot of in vitro proteasome degradation assay. HA-tr-PPM1D, HA-tr-PPM1D-K 0 , HA-fl-PPM1D, or HA-fl-PPM1D-K 0 was immunoprecipitated from HEK293T cells, the eluted PPM1D proteins were then incubated for 3 hours with recombinant human 20S proteasome with or without MG132. PSMA2 is a control to detect the proteasome. (H) Western blot of in vitro proteasome degradation assay. HA-PPM1D 400-605 was immunoprecipitated from HEK293T cells, the eluted PPM1D protein was then incubated for 3 hours with recombinant human 20S proteasome with or without MG132. PSMA2 is a control to detect the proteasome. (I) Western blot of in vitro proteasome degradation assay. Recombinant Histone H2B (top) or HA-BirA-K 0 immunoprecipitated from HEK293T cells (bottom) were incubated for 3 hours with recombinant human 20S proteasome with or without MG132. PSMA2 is a control to detect the proteasome.

Article Snippet: Cells were then collected and lysed in NP40 buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40) supplemented with 10 μM MG132, 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 78438) and 10 mM N-Ethylmaleimide (MedChemExpress, HY-D0843).

Techniques: Flow Cytometry, Expressing, Genome Wide, CRISPR, Proximity Ligation Assay, Incubation, Immunoprecipitation, Mass Spectrometry, Western Blot, In Vitro, Degradation Assay, Recombinant, Control

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) Normalized GFP:mCherry ratio detected by flow cytometry in MOLM13 cells expressing the EGFP-PPM1D- IRES-mCherry stability reporter with various C-terminal truncations treated with DMSO or MG132. Data are presented as mean ± SD (n = 3 biological replicates). (B) Schematic (left) and western blot (right) of HA-BirA-K 0 in HEK293T cells expressing either HA-BirA-K 0 or HA- BirA-K 0 -PPM1D 550-605 after treatment with MG132 or TAK-243. Asterisk indicates an unspecific band from HA antibody. (C) Western blot of Myc-PPM1D in HEK293T cells expressing Myc-fl-PPM1D, Myc-fl-PPM1D-HA, or Myc-fl- PPM1D-FLAG after treatment with cycloheximide (CHX), MG132, or TAK-243. (D) Western blot of Myc-PPM1D in HEK293T cells expressing Myc-fl-PPM1D or Myc-fl-PPM1D 1-602AAA after treatment with CHX, MG132, or TAK-243. (E) Western blot of in vitro proteasome degradation assay. HA-fl-PPM1D 1-602AAA was immunoprecipitated from HEK293T cells, the eluted PPM1D protein was then incubated for 3 hours with recombinant human 20S proteasome with or without MG132. PSMA2 is a control to detect the proteasome. (F) Western blot (top) and quantification (bottom) of a CHX chase time-course of Myc-PPM1D in HEK293T cells expressing Myc-fl-PPM1D or Myc-fl-PPM1D 1-602AAA . (G) Myc-fl-PPM1D or Myc-fl-PPM1D 1-602AAA were co-expressed with HA-Ubiquitin in HEK293T cells followed by denaturing immunoprecipitation of HA-ubiquitin and immunoblotting for Myc-PPM1D. Whole cell extract (WCE) is shown below.

Article Snippet: Cells were then collected and lysed in NP40 buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40) supplemented with 10 μM MG132, 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 78438) and 10 mM N-Ethylmaleimide (MedChemExpress, HY-D0843).

Techniques: Flow Cytometry, Expressing, Western Blot, In Vitro, Degradation Assay, Immunoprecipitation, Incubation, Recombinant, Control, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

doi: 10.64898/2026.06.01.729344

Figure Lengend Snippet: (A) Schematic (top) and results (bottom) of a ubiquitin proteasome system (UPS) targeted CRISPR screen in U937 cells expressing the EGFP-fl-PPM1D-IRES-mCherry or EGFP-tr-PPM1D-IRES-mCherry stability reporter. (B) Normalized GFP:mCherry ratio detected by flow cytometry in U937 cells expressing the EGFP-fl-PPM1D- IRES-mCherry or EGFP-tr-PPM1D-IRES-mCherry stability reporter following control or UBR5 knockdown using shRNAs. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined using unpaired two-tailed Student’s t-test; ns indicates non-significant ( p >0.05), *** p <0.001. (C) Western blot of PPM1D and UBR5 following siRNA mediated knockdown of UBR5 and treatment with cycloheximide (CHX) or MG132 in U2OS or HCT116 cells. (D) Western blot (top) and quantification (bottom) of a CHX chase time-course of V5-PPM1D in UBR5 wildtype (WT) or knockout (KO) HEK293T cells expressing either V5-tr-PPM1D (left) or V5-fl-PPM1D (right). (E) Myc-tr-PPM1D and HA-Ubiquitin were co-expressed in UBR5 WT or KO HEK293T cells with or without MG132 treatment followed by denaturing immunoprecipitation of Myc-tr-PPM1D and immunoblotting for HA- Ubiquitin. Whole cell extract (WCE) is shown below. (F) Myc-tr-PPM1D, Myc-fl-PPM1D, or Myc-PPM1D 400-605 were co-expressed with FLAG-UBR5 in HEK293T cells followed by immunoprecipitation of Myc-PPM1D and immunoblotting for UBR5. WCE is shown below. (G) Immunoblotting for tr-PPM1D following in vitro ubiquitylation assay performed by incubating recombinant tr- PPM1D and UBR5 with the necessary E1, E2, ubiquitin, and ATP substrates. (H) Western blot time-course (top) and quantification (bottom) of fl- or tr-PPM1D in U2OS parental cells following 2.5 Gy irradiation exposure.

Article Snippet: Cells were then collected and lysed in NP40 buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40) supplemented with 10 μM MG132, 1× Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 78438) and 10 mM N-Ethylmaleimide (MedChemExpress, HY-D0843).

Techniques: Ubiquitin Proteomics, CRISPR, Expressing, Flow Cytometry, Control, Knockdown, Two Tailed Test, Western Blot, Knock-Out, Immunoprecipitation, In Vitro, Ubiquitin Assay, Recombinant, Irradiation

Journal: Journal of Biological Chemistry

Article Title: The Fusion Oncoprotein PML-RARα Induces Endoplasmic Reticulum (ER)-associated Degradation of N-CoR and ER Stress

doi: 10.1074/jbc.m312121200

Figure Lengend Snippet: FIG. 4. PML-RAR induces the insolubility and ubiquitination of N-CoR. A, PML-RAR induces the insolubilty of N-CoR protein. 293T cells were transfected with the N-CoR-FLAG expression plasmid with the PML-RAR plasmid () or the empty vector (). Left, transfected cells were passively lysed, and the soluble and insoluble fractions were separated. The N-CoR protein levels were determined using anti-FLAG antibody. The same amount of lysate as that used for Western blotting was analyzed by SDS-PAGE followed by Coomassie Blue staining (lower panels). Right, transfected cells were lysed, and nuclei were isolated, which were then extracted using the same buffer described above. B, RA increases the solubility of N-CoR protein in NB4 cells. NB4 cells were treated with or without RA, and lysates were prepared using either RIPA buffer (RIPA lysate) or buffer containing salt and Nonidet P-40 (soluble fraction). The N-CoR levels were examined by Western blotting. C, PML-RAR decreases the half-life of soluble form of N-CoR. 293T cells were transfected with the N-CoR-FLAG expression plasmid with or without the PML-RAR expression plasmid, and pulse-chase-labeled. The levels of N-CoR in the soluble fraction were examined and plotted. Note that 10-fold more lysate from the PML-RAR-expressing cells was used compared with that of control lysates. D, effect of PML-RAR on the half-life of mixture of soluble and insoluble N-CoR. The experiment was performed as described in C except that the lysates were prepared using the buffer containing SDS. E, PML-RAR induces N-CoR ubiquitination. 293T cells were transfected with the plasmid expressing N-CoR-FLAG and Myc-ubiquitin with or without the PML-RAR expression plasmid, and treated with MG132. In some case, the cells were treated with RA. N-CoR-FLAG was immunoprecipitated, and the immunocomplexes were detected with the anti-Myc antibody. The filter was reprobed with anti-FLAG antibody (lower panel). F, N-CoR ubiquitination is enhanced in NB4 cells. Whole cell lysates were prepared from NB4 cells treated with the drugs indicated above, and immunoprecipitated with the anti-N-CoR antibody. Ubiquitinated N-CoR proteins (upper) and total N-CoR proteins (lower) were detected.

Article Snippet: For the ubiquitination assay of N-CoR in NB4 Cells, NB4 cells (4 107) were treated with MG132 (5 M, 12 h) or RA (1 M, 3 days), and lysates were prepared as described above, immunoprecipitated with anti-N-CoR antibody, and blotted with antiubiquitin antibody (FL-76, Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Western Blot, SDS Page, Staining, Isolation, Solubility, Pulse Chase, Labeling, Control, Immunoprecipitation

Journal: Genome Biology

Article Title: Experimental characterization of the human non-sequence-specific nucleic acid interactome

doi: 10.1186/gb-2013-14-7-r81

Figure Lengend Snippet: Validation of preferential affinity . (a) GO molecular function term significance in the various sets of proteins inferred to bind preferentially one or several subtypes of nucleic acids. We observe the clear separation between molecular functions enriched in inferred DNA- and RNA-binding proteins. Color log-scale: red = P < 1E-15, light yellow = P < 0.01, gray = P ≥ 0.01. (b) Examples of affinity preferences of selected NABPs represented by P -values in the statistical analysis (table on left) and western blots in the experimental validation (right). We note the strong agreement between preferred versus non-preferred affinities in the statistics and the blots. (C20orf72 was purified with a Myc tag in HEK293 cells instead of a specific antibody in HepG2 cells.) (c) Methylation specificity usually correlates with CG specificity, but UHRF1 and YB-1 were specific to mCG only in the statistical analysis (see reported P -values in the table on the left). Experimental validation confirmed their specificity (right); AIM2 was used as a DNA-binding non-specific control.

Article Snippet: Myc-tagged C20orf72, AIM2, UHRF1 and YB-1 were overexpressed in HEK293 cells and visualized by immunoblotting using anti-Myc-IRDye800 (Rockland Gilbertsville, PA, USA ).

Techniques: Biomarker Discovery, RNA Binding Assay, Western Blot, Purification, Methylation, Binding Assay, Control

Journal: Journal of Medicinal Chemistry

Article Title: Leveraging Targeted Protein Degradation for G Protein-Coupled Receptors: The Development of CCR2 Molecular Degraders

doi: 10.1021/acs.jmedchem.5c02920

Figure Lengend Snippet: Compound 8 induces CCR2-HiBiT degradation via the lysosomal pathway. (A) Kinetic degradation profiles of CCR2-HiBiT after treatment with compound 8 in the presence of bortezomib (10 μM), MG-132 (10 μM), MLN-4924 (250 nM), or bafilomycin-A1 (100 nM). Cells were pretreated for 2 h with proteasomal or lysosomal inhibitors before addition of compound 8 and measurement of luminescence. Kinetic profiles were obtained in a real-time HiBiT detection assay, using HEK293-LgBiT cells transiently transfected with 2 μg CCR2-HiBiT plasmid. (B) Bar graph showing the fractional RLU values after 3, 12, or 24 h of measuring luminescence. Fractional RLU values were obtained from the real-times traces shown in panel (A). Data are shown as mean ± SEM of three independent experiments performed in triplicate. Statistical differences between fractional RLU values of compound 8 in the absence and presence of proteasomal/lysosomal inhibitors at different time points were analyzed using a two-way ANOVA with Dunnett’s posthoc test: ** p < 0.01, **** p < 0.0001.

Article Snippet: Bortezomib (PS-341), MG-132, bafilomycin-A1 (Baf-A1), and chloroquine diphosphate were purchased from Selleck Chemicals (Bio-Connect, Huissen, The Netherlands); CRBN-6-5-5-VHL from MedChem Express (Bio-Connect, Huissen, The Netherlands); cycloheximide from LKT Laboratories (St. Paul, MN, USA); and chemokine ligand CCL2 from PeproTech (Cranbury, NJ, USA).

Techniques: Detection Assay, Transfection, Plasmid Preparation