Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: 14-3-3s interact with Rac1 in response to EGF in Cos-7 cells. ( A ) 14-3-3s interact with Rac1 in response to EGF in Cos-7 cells. Cos-7 cells were treated with EGF for the indicated time and the interaction between 14-3-3s and Rac1 was determined by co-immunoprecipitation (co-IP) of 14-3-3s upon immunoprecipitation (IP) of Rac1. ( B ) Sequence analysis of Rac1 reveals the presence of the mode I 14-3-3 binding motif 68 RPLSYP 73 if S71 is phosphorylated. ( C ) The highest consensus between 14-3-3 and Rac1 is within the S71-containing motif as predicted by ANN 14-3-3 Prediction software. ( D ) The effects of EGF and wortmannin on the phosphorylation of EGFR, Akt, and Rac1 S71. Cos-7 cells were treated with EGF and/or wortmannin. The phosphorylation of EGFR, Akt, and Rac S71 was revealed by immunoblotting with phospho-specific antibodies. ( E ) Quantification of the data in ( D ). The level of protein phosphorylation were quantitated by densitometry. Each value is the average of at least three experiments and the error bar is standard error. ** p < 0.01, *** p < 0.001. ( F ) The effects of EGF and wortmannin on the interaction between 14-3-3s and Rac1. Cos-7 cells were treated with EGF and/or wortmannin as indicated. The interaction between 14-3-3s and Rac1 was determined by co-IP of 14-3-3s upon IP of Rac1.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Sequencing, Binding Assay, Software, Western Blot

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: The effects of Rac1 S71 mutation to A on the interaction between 14-3-3s and Rac1 in both Cos-7 and 293T cells. ( A ) The effects of Rac1 S71 mutation to A on the interaction between 14-3-3s and Rac1 in Cos-7 cells. Cos-7 cells were transfected with wild type GFP-Rac1 and mutant GFP-S71A. The expressed Rac1 was immunoprecipitated (IPed) with an antibody to GFP, and the co-IPed 14-3-3s were examined by immunoblotting. ( B ) The effects of Rac1 S71 mutation to A on the interaction between 14-3-3s and Rac1 in Cos-7 cells by GST pulldown. The Cos-7 lysates were incubated with glutathione-agarose beads charged with GST, GST-Rac1 or mutant GST-S71A. The agarose beads were then separated and subjected to immunoblotting analysis with an antibody against 14-3-3 and GST. ( C ) The effects of Rac1 S71 mutation to A on the interaction between 14-3-3s and Rac1 in 293T cells. 293T cells were transfected with either GFP-Rac1 or GFP-S71A. GFP-Rac1 and GFP-S71A were IPed with the anti-GFP antibody. The co-IPed 14-3-3s were examined by immunoblotting.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Incubation

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: The relationship between Rac1 activity and its interaction with 14-3-3s in Cos-7 cells. ( A ) The effects of Rac1 activity on the interaction between 14-3-3s and Rac1. Cos-7 cells were transfected with either constitutively activated Rac1 (GFP-L61) or dominant-negative Rac1 (GFP-N17). The interaction of these two mutants with 14-3-3s was examined by co-IP as described above. ( B ) The effects of Rac1 S71 mutation to A on the activation of Rac1. Cos-7 cells were transfected with either GFP-Rac1 or mutant GFP-S71A, incubated with or without EGF for 15 min, and lysed. The cell lysates were incubated with GST fusion Rac-binding domain of PAK (GST-PAK) or GST bound to glutathione-agarose beads. The active Rac1 that binds to GST-PAK was determined by immunoblotting with an antibody to GFP; GST-PAK and GST were detected with an antibody to GST. ( C ) The effects of BV02 on the activation of Rac1. Cos-7 cells were transfected with GFP-Rac1 and incubated with BV02 at a concentration of 30 µM for 24 h. The cells were then incubated with or without EGF for 15 min. Cells were then lysed and the cell lysates were incubated with GST-PAK or GST bound to glutathione-agarose beads. The active Rac1 that binds to GST-PAK was determined by immunoblotting with an antibody to GFP; GST-PAK and GST were detected with an antibody to GST. ( D ) Quantification of the data in ( C ). The pulled-down active Rac1 bands were quantitated by densitometry. Each value is the average of at least three experiments and the error bar is standard error. ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Activity Assay, Transfection, Dominant Negative Mutation, Co-Immunoprecipitation Assay, Mutagenesis, Activation Assay, Incubation, Binding Assay, Western Blot, Concentration Assay

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: The interaction between Rac1 and seven 14-3-3 isoforms in Cos-7 cells. ( A ) Co-IP experiments to determine the interaction between Rac1 and seven 14-3-3 isoforms in Cos-7 cells. Cos-7 cells were transfected with GFP-Rac1. The expressed GFP-Rac1 was IPed with the anti-GFP antibody. The co-IP of various 14-3-3 isoforms was determined by immunoblotting. ( B ) Quantification of the data in ( A ). The levels of co-IPed 14-3-3s were quantitated by densitometry. Each value is the average of at least three experiments and the error bar is standard error. *** p < 0.001, ns: p > 0.1. ( C ) GST pull-down experiments to determine the interaction between three 14-3-3 isoforms including 14-3-3η, -σ, and -θ and GST-Rac1 and GST-S71A. The Cos-7 lysates were incubated with glutathione agarose beads charged with GST, GST-Rac1 or mutant GST-S71A. The agarose beads were then separated and subjected to immunoblotting analysis with antibodies against 14-3-3 η, -σ, and -θ, GST, and 14-3-3s.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Incubation, Mutagenesis

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: The interaction between Rac1 and seven 14-3-3 isoforms in 293T cells. ( A ) 293T cells were transfected with GFP-Rac1. The expressed GFP-Rac1 was IPed with the anti-GFP antibody. The co-IP of various 14-3-3 isoforms was determined by immunoblotting. ( B ) Quantification of the data in ( A ). The levels of co-IPed 14-3-3s were quantitated by densitometry. Each value is the average of at least three experiments and the error bar is standard error. * p < 0.1, *** p < 0.001.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: Disruption of the interaction between 14-3-3s and Rac1 and the effects on the subcellular localization of Rac1 and three 14-3-3 isoforms including 14-3-3η, -σ, and -θ. ( A ) The effects on the subcellular localization of Rac1. COS-7 cells were transfected with GFP-Rac1 or GFP-S71A. With or without treatment with BV02 for 24 h, the subcellular localization of GFP-Rac1 and GFP-S71A was revealed by the intrinsic fluorescence (green). The cells were counterstained with DAPI (blue). Size bar = 10 µm. ( B ) The effects on the subcellular localization of 14-3-3 isoforms. Cos-7 cells were treated with BV02 at the indicated concentration for 24 h. The cells were stained with mouse antibodies to 14-3-3η, -σ, and -θ and a rabbit antibody to α-tubulin, followed by incubation with TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG. DNA was stained by DAPI. The localization of 14-3-3 isoforms is shown as red, α-tubulin as green and chromosome/nucleus as blue. Size bar = 10 µm.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Transfection, Fluorescence, Concentration Assay, Staining, Incubation

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: The amino acid sequence alignment of human Rac1 and African green monkey Rac1.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Sequencing

Journal: Cells

Article Title: Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins

doi: 10.3390/cells8091006

Figure Lengend Snippet: Diagram to depict the interaction between Rac1 and 14-3-3s.

Article Snippet: Mouse monoclonal anti-Rac1 antibody was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques:

Journal: Journal of Virology

Article Title: Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿

doi: 10.1128/JVI.01059-09

Figure Lengend Snippet: Regulation of Nox4 by HCV core protein involves TGF-β1 autocrine signals. (A) Exogenous TGF-β1 induces the Nox4 promoter. HepG2 cells were transfected with the Nox4 promoter reporter plasmid (pGL3 −1848) with or without GFP plasmid (1.0 μg). Twenty-four hours later, transfected cells were treated with or without TGF-β1 (2 ng/ml) for 24 h. Whole-cell lysates were collected, and luciferase activity was determined by luminescence (n = 4, in triplicate). (B) Nox4 mRNA induction by TGF-β1-treated HepG2 cells. Cells were treated with or without (UT, untreated) increasing concentrations of TGF-β1 (0.5 to 2.0 ng/ml) in full-serum culture medium for 48 h. Total RNA was collected and reverse transcribed. Nox4 and GAPDH expression levels were determined by PCR amplification of total cDNA with gene-specific primers. (C) HepG2 cells were treated with TGF-β1 for 48 h as described for panel B. Cells were assayed with Diogenes reagent for superoxide generation for 45 min in the presence (+) or absence (−) of DPI (10 μM) (n = 3, in triplicate). (D) Left, HepG2 cells were transfected with either empty-vector plasmid or HCV core plasmid (1 μg). Twenty-four hours later, core-transfected cells were treated or not treated (−) with TGF-β monoclonal antibody (MAb; 2 μg/ml) for 24 h. Nox4-, core-, TGF-β1-, and GAPDH-specific PCR products were amplified from reverse-transcribed total RNA (1 μg) from cells subjected to different treatments. Right, HepG2 cells were transfected and treated as described for the left panel. Thirty-microgram amounts of total cell lysate were analyzed for protein expression by Western blotting. The immunoblot (IB) was sequentially probed with antibodies to Nox4, core, TGF-β1, and actin. Densitometry values given below blots indicate Nox4 or TGF-β protein levels relative to GAPDH protein expression. (E) HepG2 cells were transfected with empty-vector plasmid (1 μg), core plasmid (1 μg), or dominant negative TGF-βRII (ΔTβRII; 1 μg). Twenty-four hours later, cells were treated without or with TGF-β monoclonal antibody (TβMAb; 2 μg/ml) for 24 h. Cells were then assayed for superoxide production as described for panel C (n = 3). w/o DPI, without DPI. (F) HepG2 cells were transfected with empty-vector or core plasmid or cotransfected with core plasmid and dominant negative TGF-βRII (ΔTβRII) (1.0 μg each). Forty-eight hours following transfection, total RNA was collected and reverse transcribed to determine Nox4 mRNA expression by quantitative PCR (qPCR) (n = 3, in triplicate; *, P < 0.05). RLU, relative light units; Fold Δ, fold change.

Article Snippet: Western blotting was performed with the following commercially available antibodies: anti-rabbit polyclonal Nox4 antibody (Novus Biologicals, Littleton, CO), rabbit polyclonal antiactin antibody (Sigma), rabbit polyclonal anti-GAPDH (Trevigen, Gaithersburg, MD), mouse monoclonal anti-V5 (Invitrogen), goat polyclonal anti-E2 (BioDesign/Meridian Life Science, Saco, ME), goat polyclonal anti-HCV core (Pierce/ThermoScientific, Rockford, IL), mouse monoclonal anti-NS5a (Pierce/ThermoScientific), goat polyclonal anti-TGF-βRII clone (T-20), and mouse monoclonal anti-TGF-β1 (Santa Cruz) and pan-TGF-β neutralizing monoclonal antibodies (R & D Systems).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reverse Transcription, Expressing, Amplification, Western Blot, Dominant Negative Mutation, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor ?-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress ▿

doi: 10.1128/JVI.01059-09

Figure Lengend Snippet: Nox4-induced oxidative stress in response to HCV is regulated by TGF-β. HCV induces Nox4 expression and Nox4-dependent ROS generation. Expression of the HCV structural core protein upregulates endogenous TGF-β. Thus, TGF-β induces the expression of hepatic Nox4 and superoxide generation in an autocrine fashion. Inhibiting TGF-β signaling with neutralizing TGF-β-specific antibodies or an inactive form of TGF-βRII (TGF-βRII-DN, dominant negative TGF-βRII) abrogates HCV core-mediated induction of Nox4 and subsequent ROS production. MAb, monoclonal antibody.

Article Snippet: Western blotting was performed with the following commercially available antibodies: anti-rabbit polyclonal Nox4 antibody (Novus Biologicals, Littleton, CO), rabbit polyclonal antiactin antibody (Sigma), rabbit polyclonal anti-GAPDH (Trevigen, Gaithersburg, MD), mouse monoclonal anti-V5 (Invitrogen), goat polyclonal anti-E2 (BioDesign/Meridian Life Science, Saco, ME), goat polyclonal anti-HCV core (Pierce/ThermoScientific, Rockford, IL), mouse monoclonal anti-NS5a (Pierce/ThermoScientific), goat polyclonal anti-TGF-βRII clone (T-20), and mouse monoclonal anti-TGF-β1 (Santa Cruz) and pan-TGF-β neutralizing monoclonal antibodies (R & D Systems).

Techniques: Expressing, Dominant Negative Mutation

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Venn diagram of proteins identified in SW620 cell-derived sMB-R (sMV-HD), sMV-LD and Exos. b Heatmap illustration of proteins identified in sMB-R (sMV-HD), sMV-LD and Exos. Proteins present in higher abundance in sMB-R (red) as compared to sMV-LD and Exos include conserved cytokinetic proteins as well as additional cytokinetic proteins. *Proteins uniquely identified in sMB-Rs. **Proteins enriched (fold change >2) in sMB-R compared to sMV-LD and Exos. c STRING-based protein-protein interaction network analysis of 928 enriched proteins in sMB-Rs (sMV-HD) compared to sMV-LD and Exos. The interactions were “evidence”-based, with “experiments” as active interaction source and interaction threshold set at 0.900 (highest confidence). Disconnected nodes in the network are hidden. Proteins identified under biological processes or molecular processes (Gene Ontology) are indicated. Centralspindlin complex components (RACGAP1 and KIF23/MKLP1) are also indicated. d EnrichmentMap of Reactome pathways enriched in 456 proteins commonly identified in SW620 cell-derived sMB-R proteome (2300 proteins) with the proteome of MB-Rs shed by Hela cells reported recently by Peterman et al. 2019 . e Mass spectrometry-based identification of KRAS peptides (UniProtKB ID RASK_HUMAN) in sMB-Rs. Two peptides (TEYKLVVVGAGGVGK and LVVVGAGGVGK) spanning Gly-12/ Val-12 substitution in KRAS protein. Peptide spectral profiles are displayed on the right. f Immunofluorescence microscopy of SW620 cells using anti-MKLP1 and anti-KRAS G12V antibodies. Nuclei (blue) were stained with Hoechst stain. White arrows indicate the position of MB and MB-Rs. Inset represents higher magnification. Scale bar, 10 µm. g Western blot analysis of exosomes, crude 10,000 x g sMVs, and isopycnic (iodixanol-density) gradient centrifugation fractions of sMV-LD and -HD/sMB-Rs using anti-KRAS G12V antibody.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Derivative Assay, Mass Spectrometry, Immunofluorescence, Microscopy, Staining, Western Blot, Gradient Centrifugation

Journal: Communications Biology

Article Title: Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles

doi: 10.1038/s42003-021-01882-z

Figure Lengend Snippet: a Uptake of sMB-Rs by fibroblasts. Fluorescence microscopy analysis of NIH3T3 fibroblasts incubated with/without SW620 cell-derived sMB-Rs or Exos (50 µg ml −1 ) for 2 h using anti-MKLP1 and anit-RACGAP1 antibodies. b Uptake and accumulation of sMB-Rs in NIH3T3 fibroblasts was quantified by counting MKLP1 + puncta per cell; data represented as mean ± s.e.m. Nuclei (blue) were stained with Hoechst. Scale bar, 10 µm. c Internalisation of sMB-Rs by fibroblasts. Confocal microscopy of NIH3T3 fibroblasts incubated with sMB-Rs using anti-MKLP1 (in green) and anti-RAB7 (in red) antibodies. Confocal microscopy analysis along Z-axis (inset) reveal internalisation of sMB-Rs following uptake. Scale bar, 10 µm. d Intercellular transfer of sMB-R KRAS G12V into NIH3T3 cells. Fluorescence microscopy of NIH3T3 fibroblasts incubated with SW620 cell-derived sMB-Rs (5 µg) for 2 h using anti-KRAS G12V antibodies. Nuclei were stained with Hoechst stain (blue). Right panel represents fluorescence signals from left panel overlaid onto bright-field images. Inset represents enlarged image. Scale bar, 10 µm.

Article Snippet: Cells were then incubated with primary antibodies (1:100) (mouse anti-MKLP1 (Santa Cruz Biotechnology), mouse anti-RACGAP1 (Santa Cruz), rabbit anti-KRAS G12V mutant specific (Cell Signalling), rabbit anti-RAB7 (Abcam) and rabbit anti-β-tubulin (Cell Signalling) in blocking solution for 1 h at room temperature.

Techniques: Fluorescence, Microscopy, Incubation, Derivative Assay, Staining, Confocal Microscopy

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Journal of cell science

Article Title: TRPC6 channels promote dendritic growth via the CaMKIV-CREB pathway.

doi: 10.1242/jcs.026906

Figure Lengend Snippet: Fig. 5. TRPC6-induced dendritic growth depends on CaMKIV activity. (A) Total lysates of cells treated as indicated blotted with antibodies to phosphorylated CaMKIV, total CaMKIV, phosphorylated CaMKIIα or total CaMKIIα. (B) Quantification of CaMKIV phosphorylation in the neurons transfected with the indicated constructs. *P<0.05 versus ctrl. (C) Representative images of the neurons transfected at 3 DIV for 4 days with wild-type TRPC6 (WTC6) and the dominant-negative CaMKIV mutant, or shTRPC6 RNAi construct and the constitutive active CaMKIV mutant. Scale bar: 20 μm. (D,E) Quantification of total dendritic tips (D) and total dendritic length (E) of the neurons shown in C. **P<0.01 versus ctrl. All data are the means ± s.e.m. n.s., not significant.

Article Snippet: Rabbit anti-TRPC1 antibody (Alomone Labs, 1:200), rabbit anti-TRPC3 antibody (Montell, 1:500), rabbit anti-TRPC4 antibody (Alomone Labs, 1:500), rabbit antiTRPC5 antibody (Sigma, 1:200), rabbit anti-TRPC6 antibody (Alomone Labs, 1:500), mouse anti-α-tubulin antibody (Sigma, 1:1000), rabbit anti-phosphoCaMKIIα (Thr286) antibody (Santa Cruz, 1;500), mouse anti-CaMKIIα antibody (Cell Signaling, 1;1000), rabbit anti-phophoCaMKIV (Thr 196) antibody (Santa Cruz, 1:100) and rabbit anti-CaMKIV antibody (Cell Signaling, 1;500) were used as primary antibodies.

Techniques: Activity Assay, Phospho-proteomics, Transfection, Construct, Dominant Negative Mutation, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Polar interactions formed by Arg 249 with neighbouring residues in p53 WT . b Change in polar contacts with neighbouring residues due to mutation of Arg 249 to serine in p53 R249S . b Superimposed view of the mutation site in p53 WT and p53 R249S . Residues of p53 WT are shown in green and for p53 R249S they are shown in pink. Change in the structure at 249th position is highlighted by a circle. Residues that acquired significantly different conformations are shown in stick representation. c Root mean square deviations of residues in p53 WT and p53 R249S . (a) RMSD of Arg 248 during the course of MD simulation in p53 WT (green) and in p53 R249S (red). (b) RMSD of residues from 249 to 271 in p53 WT (green) and p53 R249S (red)

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Difference in the molecular surface near Y220 position in p53 WT and p53 Y220C mutant. a Size of cavity in p53 WT . b Mutation from Tyr to Cys at 220th position rearranges the conformation of surrounding residues, especially located in the loop region. Widening of the loop near 220th position and removal of the Tyr side chain creates a larger cavity in p53 Y220C protein. c Superimposition of the two molecular surfaces reveals the stringency of the p53 WT (green color) as compared to the p53 Y220C (red color). b Water network near 220th residue in p53 WT and p53 Y220C protein. a Tyr 220 stabilizes the residues of surrounding loops with the help of water molecules. b Cys 220 stabilizes the cavity by solvating it with water molecules. Binding of the withanolides with p53 WT near Tyr 220. Wi-A c and Wi-N d were found to interact with the surface residues near Tyr 220 as no deep cavity was present in p53 WT

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Interactions of Wi-A with p53 Y220C near Cys 220. a Binding pose of Wi-A within the binding site near Cys 220 b 2D representation of the interactions of Wi-A with p53 Y220C . Interactions of Wi-N with p53 Y220C protein structure. c Binding pose of Wi-N within the binding cavity of p53 Y220C near Cys 220 d 2D representation of the interactions of Wi-N with surrounding residues within the cavity of p53 Y220C . Charge complementarity of Wi-A and Wi-N with the binding cavity. Wi-A e and Wi-N f both were fitting inside the cavity according to the charge distribution within it. White region represents the hydrophobic region whereas blue and red represents the hydrophilic region

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A furnished wild type p53 function in mutant p53 (p53 Y220C ) horboring hepatoma cells. a Viability assay of human hepatocarcinoma with wild type p53 (HuH-6), mutant p53 (HuH-7), and telomerized human cells bearing p53 mutants (p53 V143A , p53 R249S and p53 R273H ). b Comparison of response of HuH-6 and HuH-7 cells to Wi-A. Dose response was observed for both the cell lines. HuH-7 showed stronger cytotoxicity to Wi-A. c Western blot showed reduction in mortalin and increase in p53 in cells treated with 1 μM Wi-A in the p53 mutants, p53 V143A and p53 R273H ; p53 R249S cells possessed low expression that remained undetected on these blots. In contrast to increase in the expression of p53 V143A and p53 R273H , mutant p53 Y220C protein expression was decreased in Wi-A treated cells. d Dose dependent decrease in mutant p53 Y220C protein expression in Wi-A treated cells. e Immunostaining of mortalin and p53 (40 x magnification) in control and Wi-A (0.5 μM) showing increase in nuclear p53 V143A and p53 R273H . HuH-6 (p53 WT ) cells showed increase in nuclear p53 staining. In contrast, HuH-7 (p53 Y220C ) cells exhibited decrease in p53 nuclear staining

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Viability Assay, Comparison, Western Blot, Expressing, Immunostaining, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced restoration of wild type p53 and induction of senescence in HuH-7 cells. a Wild type p53 reporter activity in mock (untransfected), control (transfected and untreated) and Wi-A (transfected and Wi-A treated) cells. Luciferase reporter assays driven either by p53 consensus binding sites (PG13-Luc) or by a p21 WAF-1 promoter (WWP) showed an increase in Wi-A treated cells. b Flow cytometry analysis revealed G 2 cell cycle phase arrest in HuH-7 cells. c Immunostaining of p21 WAF-1 in HuH-6 and HuH-7 control and Wi-A treated cells showing increase in p21 WAF-1 expression in the latter. d Senescence-associated β-galactosidase staining was observed in Wi-A treated HuH-6 and HuH-7 cells (10 x phase magnification). e Wi-A treated HuH-6 and HuH-7 cells showed enhanced staining for nuclear heterochromatin protein HP1γ

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Activity Assay, Control, Transfection, Luciferase, Binding Assay, Flow Cytometry, Immunostaining, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced apoptosis of HuH-7 cells. a Annexin-V staining revealed induction of early apoptosis in Wi-A treated cells. b Apoptosis in Wi-A treated HuH-7 cells was marked by cleavage of caspase3, increase in p21 WAF-1 and phosphorylated p53. c Wi-A treated cells showed Single Strand Breaks as determined by a comet assay, 40 X magnification. d Wi-A rich extract (AL-βCD) treated HuH-6 (p53 WT ) and HuH-7 (p53 Y220C ) cells showed increase and decrease in nuclear p53, respectively. e Increase in the expression of p21 WAF-1 was observed in AL-βCD treated HuH-6 and HuH-7 cells. f HuH-7 cells showed strong cytotoxicity to AL-βCD

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Staining, Single Cell Gel Electrophoresis, Expressing

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule approach for the enrichment of plasma-circulating H3-K27M-mutant nucleosomes (A) Experimental scheme: nucleosomes are extracted from HEK293 cells ectopically expressing either WT or mutant H3, and the DNA is fluorescently labeled with Cy3 by Klenow polymerase and Cy3-dATP. Next, labeled H3-K27M nucleosomes are captured on PEG-streptavidin surface covered with biotinylated antibodies targeting the H3-K27M mutant histone (α-H3-K27M) and imaged by TIRF imaging. Nucleosomes containing WT H3 do not bind the microscope coverslip and are washed prior to imaging. (B) Representative TIRF images of Cy3 signal, corresponding to surface-bound nucleosomes, extracted from HEK293 cells expressing WT or mutant histone. Nucleosomes extracted from cells expressing H3-K27M were loaded also on a surface absent of α-H3-K27M antibodies (middle), to demonstrate antibody-dependent capture. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all three images. Nucleosomes extracted from cells expressing mutant H3 show higher binding to surface coated with α-H3-K27M antibodies. (C) Quantification of the experiment described in (B). Data are presented as the mean ± SD of 50 fields of view (FOVs) per sample. (D) Experimental scheme: cfNuc from plasma are immobilized on a PEG-streptavidin covered with biotinylated α-H3-K27M antibody. Captured H3-K27M nucleosomes are detected by incubation with fluorescently labeled antibody followed by TIRF imaging. (E) Representative TIRF images of different antibodies targeting the indicated histone PTMs, while incubated on surfaces enriched for plasma-circulating H3-K27M nucleosomes (as described in D). Antibodies were tested on plasma samples from healthy individuals, as well as patients with DMG carrying WT H3 or H3-K27M. Numbers within images represent counted spots; each spot corresponds to a single surface-bound nucleosome. Scale bar applies to all images. H3K27ac and H3K36me2 antibodies are suitable for the detection of surface-bound H3-K27M nucleosomes. (F and G) Quantification of the single-molecule signal obtained for H3K27Ac antibody when incubated with and without H3-K27M enriched cfNuc from different plasma samples. Data are presented as the mean ± SD of 50 FOVs per sample.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Expressing, Labeling, Imaging, Microscopy, Binding Assay, Incubation

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule detection of mutant nucleosomes in plasma differentiates patients with H3-K27M from those with H3 WT DMG (A) Plasma samples of patients with DMG carrying WT H3 or H3-K27M mutant (light blue and dark blue, respectively) and plasma of healthy adults or children (light gray and dark gray, respectively) were analyzed as described in Figure 2 D. Mutant nucleosomes captured on surface were detected by H3K27ac antibody. Each bar represents a subject, and data are presented as the mean of the normalized H3K27ac counts ±SD of 50 FOVs per sample. (B) Boxplot representation of the data in (A), with samples grouped as follows: DMG H3-K27M ( n = 52), H3 WT glioma ( n = 16), healthy adults ( n = 21), and healthy children ( n = 29). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test and adjusted by Bonferroni correction for multiple comparisons. ∗∗ p value <0.01, ∗∗∗ p value <0.001; ns, non-significant. (C) Receiver operating characteristic (ROC) curve discriminates between plasma samples containing WT H3 nucleosomes ( n = 65) and H3-K27M-mutant nucleosomes ( n = 52) using a naive Bayes model. The area under the curve (AUC) for H3-K27M-H3K27ac normalized counts (blue line), while the red dashed diagonal line indicates expected curve for random classification. Blue shaded area represents 95% confidence interval. (D) H3-K27M-H3K27ac classifier performance; each bar represents the mean value of repeated ( n = 10,000) 4-fold cross-validation across all samples.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Detection of plasma-circulating mutant p53 in patients with DMG (A) Experimental scheme: biotinylated capture antibodies targeting total p53 protein (the WT and mutant form) are anchored to a PEG-streptavidin surface (α-p53). Plasma-circulating p53 protein molecules are captured on surface, followed by incubation with two distinct fluorescently labeled p53 antibodies: an antibody targeting all forms of p53 (“total p53,” red) and an antibody specific to the mutant conformation of p53 (“mutant p53,” green). (B) Representative TIRF images of the indicated p53 antibodies incubated on surfaces enriched for plasma-circulating p53 proteins. Scale bar applies to all images. (C) A cohort of 19 plasma samples of H3-K27M DMG patients was analyzed as described in (A) (13 samples harbor TP53 mutations and 6 harbor WT TP53). Principal component analysis (PCA) with the following input parameters: normalized counts of total p53 and mutant p53, and the ratio between mutant and total p53. Sample groups are color-coded according to known TP53 status; each dot represents one plasma sample. (D) The ratio between mutant and total p53 signal for each sample is shown. Each bar represents a sample, color-coded according to known TP53 status. Data are presented as the mean ± SD of 50 FOVs per sample. (E) Boxplot representation of the data in (D) grouped according to TP53 status (mutant p53 n = 13, WT-p53 n = 6). Boxplot limits indicate 25%–75% quantiles, the middle lines indicate the median, and the upper and lower whiskers denote the largest and smallest values, respectively, no further than 1.5× the interquartile range from the hinge. p values were calculated by Wilcoxon rank-sum exact test. ∗∗∗ p value <0.001.

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Incubation, Labeling

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet: Single-molecule measurements of mutant nucleosomes as a potential proxy for tumor growth (A) Experimental scheme for parallel measurements of cell viability and H3-K27M-mutant nucleosomes released to culture media. Cells were plated as monolayer, and a day later media was replaced to initiate the experiment. Media was collected for single-molecule measurements and replaced to account for newly released nucleosomes between examined time points. Viability measurements were conducted from a parallel culture. (B) Viability measurements and H3-K27M-H3K27ac single-molecule measurements are shown for untreated culture (left) and 5 μM ONC201-treated culture (right). The drug was replenished every 2 days. For single-molecule measurements, mean ± SD of 40 FOVs for each time point is shown. For CellTiter-Glo measurements, mean ± SE of three technical repeats for each time point is shown. (C) Clinical features, initial MRI, and MRI of disease at clinical progression of DMG patient UMPED 128. (D–J) Analysis of serial plasma samples from patients undergoing ONC201/206 treatment. Single-molecule measurements (normalized to first data point) of mutant nucleosomes (H3-K27M-H3K27ac) were performed on serial time point plasma samples plotted along with tumor cross-sectional area according to MRI. Green bars: ONC201/206 mainline treatment. Purple bars: radiation treatment. Brown bars: other treatment modalities that are detailed in Table S1 . Radiographic progression is marked with a black asterisk placed next to the corresponding MRI data point. Day 0 corresponds to the initiation of treatment. (K) Heatmap showing the correlation between the direction of change for the indicated features (see ). For the majority of patients, tumor MRI measurements show the highest correlation with the single-molecule measurements of H3-K27M-H3K27ac in plasma. The correlation was calculated based on interpolated data points to account for measurements not taken at exactly the same time. See also mutant p53 and ctDNA data in Figure S4 .

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis

Journal: Cell Reports Medicine

Article Title: Single-molecule systems for the detection and monitoring of plasma-circulating nucleosomes and oncoproteins in diffuse midline glioma

doi: 10.1016/j.xcrm.2024.101918

Figure Lengend Snippet:

Article Snippet: Histone H3 (K27M Mutant Specific) Rabbit mAb , Cell Signaling , Cat#74829; RRID: AB_2799861.

Techniques: Mutagenesis, Recombinant, Conjugation Assay, Antibody Labeling, Software