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ubiquitin e4i2j rabbit mab  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc ubiquitin e4i2j rabbit mab
    Ubiquitin E4i2j Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+ubiquitin+antibody/pm41951141-82-10-14
    Average 86 stars, based on 1 article reviews
    ubiquitin e4i2j rabbit mab - by Bioz Stars, 2026-09
    86/100 stars

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    Related Articles

    Western Blot:

    Article Title: All-Trans Retinoic Acid Modulates MicroRNA-129-5p/Hypoxia-Inducible Factor-1α/Vascular Endothelial Growth Factor Signaling and Suppresses Ets-1 in Retinal Pigment Epithelial ARPE-19 Cells.
    Article Snippet: Purpose: Proliferative vitreoretinopathy (PVR) is a vision-threatening complication of retinal detachment or ocular trauma characterized by the formation of contractile fibrotic membranes.. Retinal pigment epithelium (RPE) cells are central to PVR pathogenesis, driving maladaptive wound-healing responses.. This study investigated the effects of all-trans retinoic acid (ATRA) on RPE cell proliferation, vascular endothelial growth factor (VEGF) secretion, and miR-129-5p biogenesis, alongside the downstream regulation of Ets-1 and the hypoxiainducible factor-1a (HIF-1a)/VEGF axis.

    Article Title: The SDC1-ENO1 Axis in Cancer-Associated Fibroblasts Generates a Lactate-Rich Microenvironment that Drives Tumor Radioresistance
    Article Snippet: D ow naded rom http://aacrjournals.orgarticle-pdf/doi/10.1158/0008-5472.C AN -25-3806/35/can-25-3806.pdf by gest on 15 M arch 2026 Radiotherapy resistance remains a major barrier to effective treatment of triple-negative breast cancer (TNBC), highlighting the need to identify mechanisms driving resistance.. Here, we identified SDC1 as a pivotal mediator of cancer-associated fibroblast (CAF)-induced radioresistance in breast cancer.. SDC1 bound the TIM barrel domain of the glycolytic enzyme ENO1, preventing FBXW7-mediated degradation and driving aerobic glycolysis and lactate accumulation.

    Article Title: UVB-induced genotoxic stress activates the DNA damage response and innate immune pathways in sea urchin coelomocytes
    Article Snippet: .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro . .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro .

    Article Title: Hyperoside impairs mitochondrial respiration in chronic myeloid leukemia by promoting STUB1-mediated ubiquitination and degradation of NOX4.
    Article Snippet: Chronic myeloid leukemia (CML) cells exhibit a distinct reliance on oxidative phosphorylation (OXPHOS), presenting a metabolic vulnerability for therapeutic intervention.. While Hyperoside, a natural flavonol, demonstrates anti-leukemic activity, its precise molecular targets and impact on mitochondrial bioenergetics remain elusive.. In this study, we combined in silico modeling and biophysical validation to identify NADPH oxidase 4 (NOX4) as a direct molecular target of Hyperoside.

    Article Title: Circ_0068481 promotes pulmonary arterial smooth muscle cell remodeling via PTEN degradation and PI3K/AKT signaling activation.
    Article Snippet: Objective: Pulmonary arterial hypertension (PAH) is a progressive vascular disease characterized by pulmonary vascular remodeling and elevated pulmonary artery pressure.. Although circ_0068481, a circular RNA, has been implicated in PAH, its functional roles and underlying mechanisms remain unclear.. This study aimed to investigate the role of circ_0068481 in PAH-associated cellular dysfunction and to explore its potential molecular

    Isolation:

    Article Title: UVB-induced genotoxic stress activates the DNA damage response and innate immune pathways in sea urchin coelomocytes
    Article Snippet: .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro . .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro .

    Control:

    Article Title: UVB-induced genotoxic stress activates the DNA damage response and innate immune pathways in sea urchin coelomocytes
    Article Snippet: .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro . .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro .

    In Vitro:

    Article Title: UVB-induced genotoxic stress activates the DNA damage response and innate immune pathways in sea urchin coelomocytes
    Article Snippet: .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro . .. To further investigate this observation, western blot analysis with an anti-ubiquitin antibody (Cell Signaling Technology, #58395) was used to compare the ubiquitinated protein signal between crude protein extracts isolated from UVB-treated (1000 mJ/cm 2 ) and control coelomocytes after 6 h of recovery in vitro .

    Ubiquitin Proteomics:

    Article Title: Hyperoside impairs mitochondrial respiration in chronic myeloid leukemia by promoting STUB1-mediated ubiquitination and degradation of NOX4.
    Article Snippet: Chronic myeloid leukemia (CML) cells exhibit a distinct reliance on oxidative phosphorylation (OXPHOS), presenting a metabolic vulnerability for therapeutic intervention.. While Hyperoside, a natural flavonol, demonstrates anti-leukemic activity, its precise molecular targets and impact on mitochondrial bioenergetics remain elusive.. In this study, we combined in silico modeling and biophysical validation to identify NADPH oxidase 4 (NOX4) as a direct molecular target of Hyperoside.

    Immunoprecipitation:

    Article Title: Hyperoside impairs mitochondrial respiration in chronic myeloid leukemia by promoting STUB1-mediated ubiquitination and degradation of NOX4.
    Article Snippet: Chronic myeloid leukemia (CML) cells exhibit a distinct reliance on oxidative phosphorylation (OXPHOS), presenting a metabolic vulnerability for therapeutic intervention.. While Hyperoside, a natural flavonol, demonstrates anti-leukemic activity, its precise molecular targets and impact on mitochondrial bioenergetics remain elusive.. In this study, we combined in silico modeling and biophysical validation to identify NADPH oxidase 4 (NOX4) as a direct molecular target of Hyperoside.

    Centrifugation:

    Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma
    Article Snippet: Cell lysates were prepared using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Cat. #87788). .. After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST). .. Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

    Incubation:

    Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma
    Article Snippet: Cell lysates were prepared using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Cat. #87788). .. After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST). .. Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).

    SDS Page:

    Article Title: Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma
    Article Snippet: Cell lysates were prepared using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Cat. #87788). .. After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST). .. Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276).



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    86
    Cell Signaling Technology Inc ubiquitin e4i2j rabbit mab
    c20orf27 is a novel PP1 interactor linked to mRNA splicing that also associates separately with an E3 ubiquitin ligase complex (A) Putative RVxF motif in wild-type c20orf27 and mutation of the hydrophobic residues that should abrogate PP1 binding. Homogenous distribution of GFP-c20orf27 throughout the cytoplasm in formaldehyde-fixed U2OS cells. (B) Biofluorescence complementation (BiFC) assays confirm the re-formation of functional YFP when its N- and C-terminal halves are tagged to wild-type c20orf27 and PP1. This association is lost for the KAGA mutant. For A-B, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision CORE system using a 60x/NA1.4 oil immersion objective. (C) Quantitative IP/MS comparing GFP-c20orf27 (H) to GFP (L) in HeLa cells identified significant enrichment (>2-fold) of PP1 and a CUL2 E3 ubiquitin ligase complex with c20orf27. (D) Validation of the co-immunoprecipitation of the ligase substrate-specifier APPBP2 with GFP-c20orf27. To assess the impact of proteasomal inhibition, the assay was carried out in both untreated cells and in cells treated with the proteasome inhibitor MG132 (10 μM for 1 h; confirmed <t>by</t> <t>anti-ubiquitin</t> staining of the lysates). Ponceau S staining of the membrane (right panel) confirmed equivalent total protein loading and immunoprecipitation. (E) Functionally grouped biological process networks generated using Cytoscape:ClueGO for high-confidence hits identified in a splitPP1:c20orf27 experiment overlapped with splitPP1:RRP1B and splitPP1:TPRN. (F) 2D heatmap graph compares the distribution of protein hits between the splitPP1:NIPP1 (green) and splitPP1:c20orf27 (magenta) datasets, based on their fractional enrichment in each. Arrows indicate the threshold of 0.75 above which proteins are considered to be enriched in a particular dataset. Lines indicate spliceosome components and hnRNP proteins that were enriched above threshold for splitPP1:NIPP1 and splitPP1:c20orf27, respectively (see Table in ). (G) Accumulation of proteins biotinylated by splitPP1:c20orf27 (magenta) at nuclear speckles (arrows) stained with anti-SC35-AF488 (green). U2OS cells co-expressing NTD-c20orf27 and CTD-PP1ɣ were incubated with 50 μM biotin for 4 h prior to formaldehyde fixation and streptavidin-AF647 staining. Enlarged panels show the individual anti-SC35-AF488 and biotin signals for the boxed region. (H) Localization of a pool of GFP-c20orf27 (green) at nuclear speckles (arrows) marked by anti-SC35-AF555 staining (magenta) in methanol-fixed U2OS cells. Enlarged panels show the individual GFP-c20orf27 and anti-SC35-AF555 signals for the boxed region. Additional accumulation of GFP-c20orf27 was observed at the perinuclear region (arrowhead). (I) Analysis of co-expressed mCh-c20orf27 and APPBP2-GFP in methanol-fixed U2OS cells shows that both are enriched at the perinuclear region (arrowheads), with additional accumulation in nuclear foci (arrows) observed for mCh-c20orf27. For G–I, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision Elite system using a 60x/NA1.4 oil immersion objective. See also .
    Ubiquitin E4i2j Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PHB2-evoked NCOA4 degradation through ubiquitination. (A and B). Representative Western blotting images and quantification of NCOA4 expression in VSMCs with or without PHB2 expression (n = 6 per group, p < 0.0001). C. Quantification of relative mRNA levels of NCOA4 in VSMCs with or without PHB2 overexpression (n = 6 per group). D. Representative Western blotting images depicting the effect of proteasome inhibitor (MG132) and lysosomal inhibitors (Pepstatin A + E64d) on NCOA4 protein levels in VSMCs with or without PHB2 overexpression. (E and F) Representative Western blotting images and quantification of NCOA4 protein stability in CHX-chase assays. G. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 overexpression. H. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 silencing. Mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, no significance.

    Journal: Redox Biology

    Article Title: PHB2 ameliorates ferroptosis and aortic aneurysm/dissection through NEDD4L-dependent ubiquitination of NCOA4

    doi: 10.1016/j.redox.2026.104114

    Figure Lengend Snippet: PHB2-evoked NCOA4 degradation through ubiquitination. (A and B). Representative Western blotting images and quantification of NCOA4 expression in VSMCs with or without PHB2 expression (n = 6 per group, p < 0.0001). C. Quantification of relative mRNA levels of NCOA4 in VSMCs with or without PHB2 overexpression (n = 6 per group). D. Representative Western blotting images depicting the effect of proteasome inhibitor (MG132) and lysosomal inhibitors (Pepstatin A + E64d) on NCOA4 protein levels in VSMCs with or without PHB2 overexpression. (E and F) Representative Western blotting images and quantification of NCOA4 protein stability in CHX-chase assays. G. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 overexpression. H. Representative Co-IP images showing the ubiquitination of NCOA4 with or without PHB2 silencing. Mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, no significance.

    Article Snippet: To determine whether protein degradation was mediated by the ubiquitin proteasome pathway, cells were treated with the proteasome inhibitor MG132 (10 μM; MedChemExpress) for 6 h before harvest.

    Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Over Expression, Co-Immunoprecipitation Assay

    PHB2-induced regulation of NCOA4 ubiquitination through modulating NEDD4L activity. A. Bioinformatics prediction of interactions between NCOA4 and multiple E3 ubiquitin ligases using the UbiBrowser website ( http://ubibrowser.ncpsb.org.cn ). B. Endogenous NCOA4 and NEDD4L co-precipitated in VSMCs. C. Exogenous Flag-NCOA4 and HA-NEDD4L co-precipitated in VSMCs. D. Endogenous NCOA4 and PHB2 co-precipitated in VSMCs. E. Exogenous Flag-PHB2 and HA-NEDD4L co-precipitated in VSMCs. F. Representative Co-IP images showing that NEDD4L knockdown reduced PHB2-induced ubiquitination of NCOA4. G. Representative immunofluorescence photographs of co-localization of PHB2 and NCOA4 in VSMCs. H. Representative Western blotting images depicting subcellular localization of PHB2 and NEDD4L. I. Representative immunofluorescence photographs showing the effect of PHB2 overexpression on NEDD4L dimerization. J. Exogenous Co-IP assays of NEDD4L homodimerization via interaction between HA-NEDD4L and Flag-NEDD4L. K. Western blot analysis of NEDD4L dimer formation in vehicle versus PHB2-overexpressing groups.

    Journal: Redox Biology

    Article Title: PHB2 ameliorates ferroptosis and aortic aneurysm/dissection through NEDD4L-dependent ubiquitination of NCOA4

    doi: 10.1016/j.redox.2026.104114

    Figure Lengend Snippet: PHB2-induced regulation of NCOA4 ubiquitination through modulating NEDD4L activity. A. Bioinformatics prediction of interactions between NCOA4 and multiple E3 ubiquitin ligases using the UbiBrowser website ( http://ubibrowser.ncpsb.org.cn ). B. Endogenous NCOA4 and NEDD4L co-precipitated in VSMCs. C. Exogenous Flag-NCOA4 and HA-NEDD4L co-precipitated in VSMCs. D. Endogenous NCOA4 and PHB2 co-precipitated in VSMCs. E. Exogenous Flag-PHB2 and HA-NEDD4L co-precipitated in VSMCs. F. Representative Co-IP images showing that NEDD4L knockdown reduced PHB2-induced ubiquitination of NCOA4. G. Representative immunofluorescence photographs of co-localization of PHB2 and NCOA4 in VSMCs. H. Representative Western blotting images depicting subcellular localization of PHB2 and NEDD4L. I. Representative immunofluorescence photographs showing the effect of PHB2 overexpression on NEDD4L dimerization. J. Exogenous Co-IP assays of NEDD4L homodimerization via interaction between HA-NEDD4L and Flag-NEDD4L. K. Western blot analysis of NEDD4L dimer formation in vehicle versus PHB2-overexpressing groups.

    Article Snippet: To determine whether protein degradation was mediated by the ubiquitin proteasome pathway, cells were treated with the proteasome inhibitor MG132 (10 μM; MedChemExpress) for 6 h before harvest.

    Techniques: Ubiquitin Proteomics, Activity Assay, Co-Immunoprecipitation Assay, Knockdown, Immunofluorescence, Western Blot, Over Expression

    c20orf27 is a novel PP1 interactor linked to mRNA splicing that also associates separately with an E3 ubiquitin ligase complex (A) Putative RVxF motif in wild-type c20orf27 and mutation of the hydrophobic residues that should abrogate PP1 binding. Homogenous distribution of GFP-c20orf27 throughout the cytoplasm in formaldehyde-fixed U2OS cells. (B) Biofluorescence complementation (BiFC) assays confirm the re-formation of functional YFP when its N- and C-terminal halves are tagged to wild-type c20orf27 and PP1. This association is lost for the KAGA mutant. For A-B, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision CORE system using a 60x/NA1.4 oil immersion objective. (C) Quantitative IP/MS comparing GFP-c20orf27 (H) to GFP (L) in HeLa cells identified significant enrichment (>2-fold) of PP1 and a CUL2 E3 ubiquitin ligase complex with c20orf27. (D) Validation of the co-immunoprecipitation of the ligase substrate-specifier APPBP2 with GFP-c20orf27. To assess the impact of proteasomal inhibition, the assay was carried out in both untreated cells and in cells treated with the proteasome inhibitor MG132 (10 μM for 1 h; confirmed by anti-ubiquitin staining of the lysates). Ponceau S staining of the membrane (right panel) confirmed equivalent total protein loading and immunoprecipitation. (E) Functionally grouped biological process networks generated using Cytoscape:ClueGO for high-confidence hits identified in a splitPP1:c20orf27 experiment overlapped with splitPP1:RRP1B and splitPP1:TPRN. (F) 2D heatmap graph compares the distribution of protein hits between the splitPP1:NIPP1 (green) and splitPP1:c20orf27 (magenta) datasets, based on their fractional enrichment in each. Arrows indicate the threshold of 0.75 above which proteins are considered to be enriched in a particular dataset. Lines indicate spliceosome components and hnRNP proteins that were enriched above threshold for splitPP1:NIPP1 and splitPP1:c20orf27, respectively (see Table in ). (G) Accumulation of proteins biotinylated by splitPP1:c20orf27 (magenta) at nuclear speckles (arrows) stained with anti-SC35-AF488 (green). U2OS cells co-expressing NTD-c20orf27 and CTD-PP1ɣ were incubated with 50 μM biotin for 4 h prior to formaldehyde fixation and streptavidin-AF647 staining. Enlarged panels show the individual anti-SC35-AF488 and biotin signals for the boxed region. (H) Localization of a pool of GFP-c20orf27 (green) at nuclear speckles (arrows) marked by anti-SC35-AF555 staining (magenta) in methanol-fixed U2OS cells. Enlarged panels show the individual GFP-c20orf27 and anti-SC35-AF555 signals for the boxed region. Additional accumulation of GFP-c20orf27 was observed at the perinuclear region (arrowhead). (I) Analysis of co-expressed mCh-c20orf27 and APPBP2-GFP in methanol-fixed U2OS cells shows that both are enriched at the perinuclear region (arrowheads), with additional accumulation in nuclear foci (arrows) observed for mCh-c20orf27. For G–I, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision Elite system using a 60x/NA1.4 oil immersion objective. See also .

    Journal: iScience

    Article Title: SplitTurboID mapping of dimeric protein phosphatase complex interactomes

    doi: 10.1016/j.isci.2026.115195

    Figure Lengend Snippet: c20orf27 is a novel PP1 interactor linked to mRNA splicing that also associates separately with an E3 ubiquitin ligase complex (A) Putative RVxF motif in wild-type c20orf27 and mutation of the hydrophobic residues that should abrogate PP1 binding. Homogenous distribution of GFP-c20orf27 throughout the cytoplasm in formaldehyde-fixed U2OS cells. (B) Biofluorescence complementation (BiFC) assays confirm the re-formation of functional YFP when its N- and C-terminal halves are tagged to wild-type c20orf27 and PP1. This association is lost for the KAGA mutant. For A-B, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision CORE system using a 60x/NA1.4 oil immersion objective. (C) Quantitative IP/MS comparing GFP-c20orf27 (H) to GFP (L) in HeLa cells identified significant enrichment (>2-fold) of PP1 and a CUL2 E3 ubiquitin ligase complex with c20orf27. (D) Validation of the co-immunoprecipitation of the ligase substrate-specifier APPBP2 with GFP-c20orf27. To assess the impact of proteasomal inhibition, the assay was carried out in both untreated cells and in cells treated with the proteasome inhibitor MG132 (10 μM for 1 h; confirmed by anti-ubiquitin staining of the lysates). Ponceau S staining of the membrane (right panel) confirmed equivalent total protein loading and immunoprecipitation. (E) Functionally grouped biological process networks generated using Cytoscape:ClueGO for high-confidence hits identified in a splitPP1:c20orf27 experiment overlapped with splitPP1:RRP1B and splitPP1:TPRN. (F) 2D heatmap graph compares the distribution of protein hits between the splitPP1:NIPP1 (green) and splitPP1:c20orf27 (magenta) datasets, based on their fractional enrichment in each. Arrows indicate the threshold of 0.75 above which proteins are considered to be enriched in a particular dataset. Lines indicate spliceosome components and hnRNP proteins that were enriched above threshold for splitPP1:NIPP1 and splitPP1:c20orf27, respectively (see Table in ). (G) Accumulation of proteins biotinylated by splitPP1:c20orf27 (magenta) at nuclear speckles (arrows) stained with anti-SC35-AF488 (green). U2OS cells co-expressing NTD-c20orf27 and CTD-PP1ɣ were incubated with 50 μM biotin for 4 h prior to formaldehyde fixation and streptavidin-AF647 staining. Enlarged panels show the individual anti-SC35-AF488 and biotin signals for the boxed region. (H) Localization of a pool of GFP-c20orf27 (green) at nuclear speckles (arrows) marked by anti-SC35-AF555 staining (magenta) in methanol-fixed U2OS cells. Enlarged panels show the individual GFP-c20orf27 and anti-SC35-AF555 signals for the boxed region. Additional accumulation of GFP-c20orf27 was observed at the perinuclear region (arrowhead). (I) Analysis of co-expressed mCh-c20orf27 and APPBP2-GFP in methanol-fixed U2OS cells shows that both are enriched at the perinuclear region (arrowheads), with additional accumulation in nuclear foci (arrows) observed for mCh-c20orf27. For G–I, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision Elite system using a 60x/NA1.4 oil immersion objective. See also .

    Article Snippet: Monoclonal mouse anti-Ubiquitin , Santa Cruz , Cat#sc-8017; RRID: AB_628423.

    Techniques: Ubiquitin Proteomics, Mutagenesis, Binding Assay, Functional Assay, Staining, Protein-Protein interactions, Biomarker Discovery, Immunoprecipitation, Inhibition, Membrane, Generated, Expressing, Incubation