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: Cancer Research

Article Title: Breast Cancer Migration and Invasion Depend on Proteasome Degradation of Regulator of G-Protein Signaling 4

doi: 10.1158/0008-5472.can-08-3564

Figure Lengend Snippet: Figure 6. Endogenous RGS4 protein inhibits breast cancer cell migration and invasion. Columns, mean; bars, SE with *, P < 0.01 and **, P < 0.001. A, quantitative real-time PCR analysis of RGS4 mRNA levels (n = 3). Inset, representative Western blot (n = 5) of RGS4 protein levels. MDA-MB-231 cell lysate plus 0.25 ng purified RGS4 protein as positive control. MDA-MB-231 cells (B) and MDA-MB-436 cells (C) treated for 4 h with or without MG132 (20 Amol/L) F cycloheximide (CHX; 0.1 mmol/L; n = 5) before Western blot analysis (insets) or Transwell migration and invasion assays. D, silencing endogenous RGS4 expression reverses the proteasome blockade-induced inhibition of MDA-MB-231 cell migration and invasion. Cells stably expressing RGS4 shRNA or GFP shRNA were treated with or without MG132 (n = 3) and then subjected to Western blot analysis (top) or Transwell migration and invasion assays (bottom). Untreated cells expressing GFP shRNA were set as 100%.

Article Snippet: Cell-permeable C3 transferase and F-actin visualization kit (Cytoskeleton); NSC23766, MG132, PSI, and chloroquine (Calbiochem); epidermal growth factor (EGF) and fibronectin (BD Biosciences); CXCL12 (R&D Systems); BMS-200261 and TFLLR (Peptides International); SCH79797 (Tocris); cycloheximide (Sigma); rabbit anti-PAR1 antibody (Santa Cruz Biotechnology); and monoclonal anti-hemagglutinin (HA) antibody (Covance).

Techniques: Migration, Real-time Polymerase Chain Reaction, Western Blot, Purification, Positive Control, Expressing, Inhibition, Stable Transfection, shRNA

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 1. Hypoxia regulates GCM1 degradation. A, both GCM1 transcript and protein levels are decreased in placental cells under hypoxia. Total RNA was purified from BeWo, BeWo31, and JAR cells under normoxia (white bars) or hypoxia (black bars) for 48 h. One g of RNA was converted into the first strand cDNA using oligo(dT)20 as primer, followed by quantitative real time PCR. Mean values and S.D. of the ratio of GCM1 to -actin RNA copy number are shown (left). Cell protein extracts from BeWo cells under normoxia or hypoxia for 48 h were immunoblotted with GCM1 or -tubulin antibody (right). B, hypoxia regulates GCM1 expression at the post-translational level. BeWo31 cells were cultured under hypoxia for 48 h or exposed to 250 M CoCl2 for 36 or 48 h. Cells were then harvested for immunoblotting with HA or -tubulin antibody (left) or for RT-PCR analysis of the transcript levels of HA-GCM1 and glyceraldehyde-3-phosphate dehydrogenase. C, the effect of hypoxia on GCM1 protein level is reversible. BeWo31 cells were mock-treated or pretreated with 250 M CoCl2 for 24 h. The medium was then replaced with fresh medium without CoCl2, and culture was continued for the indicated period of time before harvesting the cells for immunoblotting with HA or -actin antibody. D, proteasome is involved in the decreased GCM1 protein level induced by hypoxia. BeWo31 cells were mock-treated or treated with 250 M CoCl2 in the presence or absence of 40 M MG132 for 24 h. Cells were then harvested for immunoblotting with HA or -actin antibody.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Purification, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 3. GSK-3 controls GCM1 ubiquitination and degradation. A, GCM1 ubiquitination is regulated by GSK-3. 293T cells were transfected with 1 g of pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid. At 24 h post-transfection, cells were treated with 40 M MG132 for an additional 4 h and thensubjecttoubiquitinationanalysisbyimmunoprecipitation(IP)withFLAGmAbandimmunoblotting(IB)withHAmAb(left).Inaseparateexperiment,293T cellsweretransfectedwith1gofpGCM1-FLAGandpHA-Ubplus10nMGL2orGSK-3siRNAandincubatedasaboveforubiquitinationanalysis.B,thehalf-life of GCM1 is prolonged by inhibition of GSK-3. 293T cells were transfected with 0.35 g of pGCM1-FLAG. At 24 h post-transfection, cells were pretreated with or without 50 mM LiCl for 2 h before incubation with 75 M cycloheximide for the indicated period of time. In a separate experiment, 293T cells were transfected with0.35gofpGCM1-FLAGplus10nMGL2orGSK-3siRNA.At24hpost-transfection,cellswereincubatedwith75Mcycloheximidefortheindicatedperiod of time. Cells were then harvested for immunoblotting with FLAG or -tubulin antibody.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Inhibition, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 4. Identification of GSK-3 phosphorylation sites and a C-terminal destruction motif in GCM1. A, Ser322 and Ser326 are required for GCM1 ubiquitination. 293T cells were transfected with 1 g of pHA-Ub and wild-type or mutant pGCM1-FLAG, treated with MG132, and subject to ubiquitination analysis as described in the legend to Fig. 3A. B, Ser322 and Ser326 are involved in regulation of GCM1 stability. 293T cells were transfected with the indicated pGCM1-FLAG expression plasmid for protein stability analysis as described in the legend to Fig. 3B. C and D, FBW2 interacts with the C-terminal TpSWPCpS (where pS represents phosphoserine) destruction motif in GCM1. GST- or GST-FBW2-loaded glutathione beads were incubated with 100 g of the cell lysate prepared from 293T cells transfected with wild-type or mutant pGCM1-FLAG expression plasmids for pull-down analysis, followed by immunoblotting with FLAG mAb. Biotinylated unmodified peptides or phosphopeptides covering amino acids 313–333 were incubated with 100 g of cell lysate prepared from 293T cells transfected with pFBW2-Myc, pTrcp-Myc, or pSKP2-Myc for pull-down analysis, followed by immunoblotting with Myc mAb. IP, immunoprecipi- tation; IB, immunoblot.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Hypoxia-induced GCM1 Degradation

doi: 10.1074/jbc.m109.016170

Figure Lengend Snippet: FIGURE 5. Hypoxia induces Ser322 phosphorylation in GCM1 in vivo. A, GSK-3 phosphorylates Ser322 under hypoxia. 293T cells were transfected with pHA-GCM1 plus GL2 or GSK-3 siRNA. The transfected cells were subjected to hypoxic conditions for 24 h in the presence of MG132, followed by immunoprecipitation (IP) with HA mAb and immunoblotting (IB) with Ser(P)322-GCM1 Ab. B, Ser322 phosphorylation in placental cells under hypoxia. BeWo31 cells were cultured under normoxia or hypoxia for 48 h and mock-treated or treated with the indicated combination of MG132 and LiCl, followed by immunoprecipitation with HA mAb and immunoblot- ting with HA, Ser(P)322-GCM1, or Ser(P)326-GCM1 antibody. C, GCM1 degradation under placental hypoxia contributes to the pathogenesis of preeclampsia.

Article Snippet: Immunoprecipitation and Immunoblotting—For in vivo ubiquitination assays of GCM1, 293T cells were transfected with pGCM1-FLAG, pHA-Ub, and the indicated GSK-3 expression plasmid or siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by treatment with MG132.

Techniques: Phospho-proteomics, In Vivo, Transfection, Immunoprecipitation, Western Blot, Cell Culture

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