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    Addgene inc pcdna3 1 mgreenlantern
    Pcdna3 1 Mgreenlantern, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 4. SERBP1 interacts with <t>PARP1</t> and influences PARylation. (A) Results of IP-western in U251 cells with control and anti-SERBP1 antibodies confirm SERBP1 interaction with PARP1, NCL, and SYNCRIP. SERBP1 and PARP1 strong association is corroborated by their high expression correlation in different studies (B) and similar expression profiles during cortex development according to Cortecon (van de Leemput et al., 2014). (C). (D) PARylation sites observed in SERBP1 protein according to Gibson et al., 2016; Martello et al., 2016. (E) PARP1 ADP-ribosylates SERBP1 in vitro. Purified recombinant PARP1 (0.1 µM) and SERBP1 (0.6 µM) were combined in a reaction with or without sheared salmon sperm DNA (sssDNA) (100 ng/ µL) and NAD+ (100 µM) as indicated. The reaction products were analyzed by SDS-PAGE with silver staining (left) and Western blotting for PAR (right). Uncropped gels are shown. (F) Venn diagram shows that majority of SERBP1-associated factors get PARylated and/or bind PAR (Gibson et al., 2016; Martello et al., 2016; Dasovich et al., 2021). (G) Increase of PARylation levels in 293T and U251 GBM cells after H2O2 treatment. (H) SBP-SERBP1 and Flag-PARP1 were co-transfected into 293T cells. Cells were treated with H2O2 to induce PARylation and a pull-down experiment with streptavidin beads was performed. Western analysis showed increased SERBP1 association with PARP1 in cells treated with H2O2. SBP-SERBP1-His detected by His antibody; Flag-PARP1 detected by Flag antibody. (I) SBP-SERBP1 was transfected into 293T cells. Cells were treated with DMSO or 10 μM PJ34 (PARP inhibitor) for two hours and a pull-down experiment with streptavidin beads was performed. Western analysis showed a decrease in SERBP1 association with PARP1 and GPBP1. (J) SERBP1 transgenic expression (mGreen-SERBP1) in 293T cells increased the levels of PARylated proteins as indicated by PAR-detecting agent. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1, Supplementary file 5 and Supplementary file 6.
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    Figure 4. SERBP1 interacts with <t>PARP1</t> and influences PARylation. (A) Results of IP-western in U251 cells with control and anti-SERBP1 antibodies confirm SERBP1 interaction with PARP1, NCL, and SYNCRIP. SERBP1 and PARP1 strong association is corroborated by their high expression correlation in different studies (B) and similar expression profiles during cortex development according to Cortecon (van de Leemput et al., 2014). (C). (D) PARylation sites observed in SERBP1 protein according to Gibson et al., 2016; Martello et al., 2016. (E) PARP1 ADP-ribosylates SERBP1 in vitro. Purified recombinant PARP1 (0.1 µM) and SERBP1 (0.6 µM) were combined in a reaction with or without sheared salmon sperm DNA (sssDNA) (100 ng/ µL) and NAD+ (100 µM) as indicated. The reaction products were analyzed by SDS-PAGE with silver staining (left) and Western blotting for PAR (right). Uncropped gels are shown. (F) Venn diagram shows that majority of SERBP1-associated factors get PARylated and/or bind PAR (Gibson et al., 2016; Martello et al., 2016; Dasovich et al., 2021). (G) Increase of PARylation levels in 293T and U251 GBM cells after H2O2 treatment. (H) SBP-SERBP1 and Flag-PARP1 were co-transfected into 293T cells. Cells were treated with H2O2 to induce PARylation and a pull-down experiment with streptavidin beads was performed. Western analysis showed increased SERBP1 association with PARP1 in cells treated with H2O2. SBP-SERBP1-His detected by His antibody; Flag-PARP1 detected by Flag antibody. (I) SBP-SERBP1 was transfected into 293T cells. Cells were treated with DMSO or 10 μM PJ34 (PARP inhibitor) for two hours and a pull-down experiment with streptavidin beads was performed. Western analysis showed a decrease in SERBP1 association with PARP1 and GPBP1. (J) SERBP1 transgenic expression (mGreen-SERBP1) in 293T cells increased the levels of PARylated proteins as indicated by PAR-detecting agent. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1, Supplementary file 5 and Supplementary file 6.
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    Journal: eLife

    Article Title: SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis

    doi: 10.7554/eLife.98152

    Figure Lengend Snippet:

    Article Snippet: SERBP1 ORF was inserted into the pcDNA3.1-mGreenLantern plasmid (RRID: Addgene_1619122 ) to make mGreen-SERBP1.

    Techniques: Transfection, Construct, Control, Binding Assay, Recombinant, Plasmid Preparation, Clone Assay, In Situ, Drug discovery, Concentration Assay, Software, Comparison, Microscopy

    Figure 4. SERBP1 interacts with PARP1 and influences PARylation. (A) Results of IP-western in U251 cells with control and anti-SERBP1 antibodies confirm SERBP1 interaction with PARP1, NCL, and SYNCRIP. SERBP1 and PARP1 strong association is corroborated by their high expression correlation in different studies (B) and similar expression profiles during cortex development according to Cortecon (van de Leemput et al., 2014). (C). (D) PARylation sites observed in SERBP1 protein according to Gibson et al., 2016; Martello et al., 2016. (E) PARP1 ADP-ribosylates SERBP1 in vitro. Purified recombinant PARP1 (0.1 µM) and SERBP1 (0.6 µM) were combined in a reaction with or without sheared salmon sperm DNA (sssDNA) (100 ng/ µL) and NAD+ (100 µM) as indicated. The reaction products were analyzed by SDS-PAGE with silver staining (left) and Western blotting for PAR (right). Uncropped gels are shown. (F) Venn diagram shows that majority of SERBP1-associated factors get PARylated and/or bind PAR (Gibson et al., 2016; Martello et al., 2016; Dasovich et al., 2021). (G) Increase of PARylation levels in 293T and U251 GBM cells after H2O2 treatment. (H) SBP-SERBP1 and Flag-PARP1 were co-transfected into 293T cells. Cells were treated with H2O2 to induce PARylation and a pull-down experiment with streptavidin beads was performed. Western analysis showed increased SERBP1 association with PARP1 in cells treated with H2O2. SBP-SERBP1-His detected by His antibody; Flag-PARP1 detected by Flag antibody. (I) SBP-SERBP1 was transfected into 293T cells. Cells were treated with DMSO or 10 μM PJ34 (PARP inhibitor) for two hours and a pull-down experiment with streptavidin beads was performed. Western analysis showed a decrease in SERBP1 association with PARP1 and GPBP1. (J) SERBP1 transgenic expression (mGreen-SERBP1) in 293T cells increased the levels of PARylated proteins as indicated by PAR-detecting agent. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1, Supplementary file 5 and Supplementary file 6.

    Journal: eLife

    Article Title: SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis

    doi: 10.7554/elife.98152

    Figure Lengend Snippet: Figure 4. SERBP1 interacts with PARP1 and influences PARylation. (A) Results of IP-western in U251 cells with control and anti-SERBP1 antibodies confirm SERBP1 interaction with PARP1, NCL, and SYNCRIP. SERBP1 and PARP1 strong association is corroborated by their high expression correlation in different studies (B) and similar expression profiles during cortex development according to Cortecon (van de Leemput et al., 2014). (C). (D) PARylation sites observed in SERBP1 protein according to Gibson et al., 2016; Martello et al., 2016. (E) PARP1 ADP-ribosylates SERBP1 in vitro. Purified recombinant PARP1 (0.1 µM) and SERBP1 (0.6 µM) were combined in a reaction with or without sheared salmon sperm DNA (sssDNA) (100 ng/ µL) and NAD+ (100 µM) as indicated. The reaction products were analyzed by SDS-PAGE with silver staining (left) and Western blotting for PAR (right). Uncropped gels are shown. (F) Venn diagram shows that majority of SERBP1-associated factors get PARylated and/or bind PAR (Gibson et al., 2016; Martello et al., 2016; Dasovich et al., 2021). (G) Increase of PARylation levels in 293T and U251 GBM cells after H2O2 treatment. (H) SBP-SERBP1 and Flag-PARP1 were co-transfected into 293T cells. Cells were treated with H2O2 to induce PARylation and a pull-down experiment with streptavidin beads was performed. Western analysis showed increased SERBP1 association with PARP1 in cells treated with H2O2. SBP-SERBP1-His detected by His antibody; Flag-PARP1 detected by Flag antibody. (I) SBP-SERBP1 was transfected into 293T cells. Cells were treated with DMSO or 10 μM PJ34 (PARP inhibitor) for two hours and a pull-down experiment with streptavidin beads was performed. Western analysis showed a decrease in SERBP1 association with PARP1 and GPBP1. (J) SERBP1 transgenic expression (mGreen-SERBP1) in 293T cells increased the levels of PARylated proteins as indicated by PAR-detecting agent. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1, Supplementary file 5 and Supplementary file 6.

    Article Snippet: DOI: https://doi.org/10.7554/eLife.98152 20 of 37 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent mGreen- SERBP1 (plasmid) This paper SERBP1 ORF cloned in frame in pcDNA3.1- mGreenLantern backbone Recombinant DNA reagent Flag- PARP1 Addgene RRID:Addgene_111575 Peptide, recombinant protein 6xHis- tagged SERBP1 Baudin et al., 2021 Commercial assay or kit Lipofectamine RNAiMAX Invitrogen Cat#:13778150 Commercial assay or kit Streptavidin beads GE Healthcare Life Sciences Cat#:17- 5113- 01 Commercial assay or kit Cell Titer Glo 2.0 Promega Cat#:G9243 Commercial assay or kit Duolink PLA in Situ Red starter kit mouse/rabbit Sigma- Aldrich Cat#:DUO92101 Chemical compound, drug Cambridge Cancer Compound Library Selleck Chem Cat#:L2300 100 nM in 0.1% DMSO treatment concentration Chemical compound, drug Puromycin Sigma- Aldrich Cat#:P7255 Chemical compound, drug Paclitaxel Cayman Chem Cat#:10461 Chemical compound, drug PARP inhibitor PJ34 Enzo Cat#:ALX- 270–289 Software, algorithm Mascot v2.7.0 Matrix Science RRID:SCR_014322 Software, algorithm Scaffold v4.9.0 Proteome Software RRID:SCR_014321 Software, algorithm ImageJ FIJI NIH RRID:SCR_002285 Software, algorithm BioInfoRx https://bioinforx.com/ apps/venn.php Software, algorithm Nematode genome comparison browser http://nemates.org/MA/ progs/overlap_stats.html Software, algorithm ShinyGO v0.67 & v0.77 Ge et al., 2020 Software, algorithm Metascape v3.5 Zhou et al., 2019 Software, algorithm Panther v17.0 Thomas et al., 2022 Software, algorithm Revigo Supek et al., 2011 Software, algorithm STRING v11.5 Szklarczyk et al., 2023 Software, algorithm Cytoscape Doncheva et al., 2023 Software, algorithm R2 http://r2.amc.nl Software, algorithm dcGO Enrichment mining service Fang and Gough, 2013 Software, algorithm STAR v2.7.7.a Dobin et al., 2013 Software, algorithm rMATS v4.1.2 Shen et al., 2014 Software, algorithm rmats2sashimiplot tool https://github.com/ Xinglab/rmats2sashimiplot; Xinglab, 2024 Continued Continued on next page Breunig, Lei, Montalbano et al. eLife 2024;13:RP98152.

    Techniques: Western Blot, Control, Expressing, In Vitro, Purification, Recombinant, SDS Page, Silver Staining, Transfection, Transgenic Assay

    Figure 5. Shared SERBP1 and PARP1 interactors. (A) Venn diagram shows the overlap between PARP1 (Mosler et al., 2022) and SERBP1 interactomes. Analysis of shared interactors indicated that the majority of them are PAR binding (Dasovich et al., 2021) and/or get PARylated (Gibson et al., 2016; Martello et al., 2016). (B) Selection of enriched GO terms (biological processes) related to SERBP1-PARP1 shared interactors according to ShinyGO (Ge et al., 2020). FE = fold enrichment; FDR = false discovery rate. (C) Network showing shared SERBP1 and PARP1 interactors implicated in ribosome biogenesis. (D) Proposed SERBP1-PARP1 feedback model; SERBP1 function and association with partner proteins is modulated by PARylation while SERBP1 influences PARP activity. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1 and Supplementary file 6.

    Journal: eLife

    Article Title: SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis

    doi: 10.7554/elife.98152

    Figure Lengend Snippet: Figure 5. Shared SERBP1 and PARP1 interactors. (A) Venn diagram shows the overlap between PARP1 (Mosler et al., 2022) and SERBP1 interactomes. Analysis of shared interactors indicated that the majority of them are PAR binding (Dasovich et al., 2021) and/or get PARylated (Gibson et al., 2016; Martello et al., 2016). (B) Selection of enriched GO terms (biological processes) related to SERBP1-PARP1 shared interactors according to ShinyGO (Ge et al., 2020). FE = fold enrichment; FDR = false discovery rate. (C) Network showing shared SERBP1 and PARP1 interactors implicated in ribosome biogenesis. (D) Proposed SERBP1-PARP1 feedback model; SERBP1 function and association with partner proteins is modulated by PARylation while SERBP1 influences PARP activity. Datasets used to prepare the figure, and detailed analyses are in Supplementary file 1 and Supplementary file 6.

    Article Snippet: DOI: https://doi.org/10.7554/eLife.98152 20 of 37 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent mGreen- SERBP1 (plasmid) This paper SERBP1 ORF cloned in frame in pcDNA3.1- mGreenLantern backbone Recombinant DNA reagent Flag- PARP1 Addgene RRID:Addgene_111575 Peptide, recombinant protein 6xHis- tagged SERBP1 Baudin et al., 2021 Commercial assay or kit Lipofectamine RNAiMAX Invitrogen Cat#:13778150 Commercial assay or kit Streptavidin beads GE Healthcare Life Sciences Cat#:17- 5113- 01 Commercial assay or kit Cell Titer Glo 2.0 Promega Cat#:G9243 Commercial assay or kit Duolink PLA in Situ Red starter kit mouse/rabbit Sigma- Aldrich Cat#:DUO92101 Chemical compound, drug Cambridge Cancer Compound Library Selleck Chem Cat#:L2300 100 nM in 0.1% DMSO treatment concentration Chemical compound, drug Puromycin Sigma- Aldrich Cat#:P7255 Chemical compound, drug Paclitaxel Cayman Chem Cat#:10461 Chemical compound, drug PARP inhibitor PJ34 Enzo Cat#:ALX- 270–289 Software, algorithm Mascot v2.7.0 Matrix Science RRID:SCR_014322 Software, algorithm Scaffold v4.9.0 Proteome Software RRID:SCR_014321 Software, algorithm ImageJ FIJI NIH RRID:SCR_002285 Software, algorithm BioInfoRx https://bioinforx.com/ apps/venn.php Software, algorithm Nematode genome comparison browser http://nemates.org/MA/ progs/overlap_stats.html Software, algorithm ShinyGO v0.67 & v0.77 Ge et al., 2020 Software, algorithm Metascape v3.5 Zhou et al., 2019 Software, algorithm Panther v17.0 Thomas et al., 2022 Software, algorithm Revigo Supek et al., 2011 Software, algorithm STRING v11.5 Szklarczyk et al., 2023 Software, algorithm Cytoscape Doncheva et al., 2023 Software, algorithm R2 http://r2.amc.nl Software, algorithm dcGO Enrichment mining service Fang and Gough, 2013 Software, algorithm STAR v2.7.7.a Dobin et al., 2013 Software, algorithm rMATS v4.1.2 Shen et al., 2014 Software, algorithm rmats2sashimiplot tool https://github.com/ Xinglab/rmats2sashimiplot; Xinglab, 2024 Continued Continued on next page Breunig, Lei, Montalbano et al. eLife 2024;13:RP98152.

    Techniques: Binding Assay, Selection, Activity Assay

    Figure 8. SERBP1 association with PARP1 in Alzheimer’s brains. (A) Representative co-immunofluorescence of SERBP1 and PARP1 in AD and age- matched control brain tissues. PARP1 and SERBP1 are represented in gray while merged images represent PARP1 (red), SERBP1 (green). DAPI was used to stain nuclei (blue). Magnification ×20 and white scale bar: 100 µm. Each frame has a zoomed inset representing the detailed distribution of each target. (B) Pearson Coefficient (PCC) of co-localizing SERBP1 and PARP1 between cells of age-matched control and AD brains (CTR vs. AD, **** p<0.001 paired t-test). (C) Fluorescence intensity profiles of PARP1 (red), SERBP1 (green), and DAPI (blue) in representative cells from age-matched control and AD brains. Distance is represented in pixels and fluorescence intensity as Grey value obtained using ImageJ FIJI software. (D) Representative PLA of SERBP1 vs. PARP1 in AD and aged-matched control brains (magnification 40 x and white scale bar: 50 µm). Percentage of positive area to PLA fluorescence in region of interests in AD and control brains (Ctr vs AD, ** p<0.01, paired t-test).

    Journal: eLife

    Article Title: SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis

    doi: 10.7554/elife.98152

    Figure Lengend Snippet: Figure 8. SERBP1 association with PARP1 in Alzheimer’s brains. (A) Representative co-immunofluorescence of SERBP1 and PARP1 in AD and age- matched control brain tissues. PARP1 and SERBP1 are represented in gray while merged images represent PARP1 (red), SERBP1 (green). DAPI was used to stain nuclei (blue). Magnification ×20 and white scale bar: 100 µm. Each frame has a zoomed inset representing the detailed distribution of each target. (B) Pearson Coefficient (PCC) of co-localizing SERBP1 and PARP1 between cells of age-matched control and AD brains (CTR vs. AD, **** p<0.001 paired t-test). (C) Fluorescence intensity profiles of PARP1 (red), SERBP1 (green), and DAPI (blue) in representative cells from age-matched control and AD brains. Distance is represented in pixels and fluorescence intensity as Grey value obtained using ImageJ FIJI software. (D) Representative PLA of SERBP1 vs. PARP1 in AD and aged-matched control brains (magnification 40 x and white scale bar: 50 µm). Percentage of positive area to PLA fluorescence in region of interests in AD and control brains (Ctr vs AD, ** p<0.01, paired t-test).

    Article Snippet: DOI: https://doi.org/10.7554/eLife.98152 20 of 37 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent mGreen- SERBP1 (plasmid) This paper SERBP1 ORF cloned in frame in pcDNA3.1- mGreenLantern backbone Recombinant DNA reagent Flag- PARP1 Addgene RRID:Addgene_111575 Peptide, recombinant protein 6xHis- tagged SERBP1 Baudin et al., 2021 Commercial assay or kit Lipofectamine RNAiMAX Invitrogen Cat#:13778150 Commercial assay or kit Streptavidin beads GE Healthcare Life Sciences Cat#:17- 5113- 01 Commercial assay or kit Cell Titer Glo 2.0 Promega Cat#:G9243 Commercial assay or kit Duolink PLA in Situ Red starter kit mouse/rabbit Sigma- Aldrich Cat#:DUO92101 Chemical compound, drug Cambridge Cancer Compound Library Selleck Chem Cat#:L2300 100 nM in 0.1% DMSO treatment concentration Chemical compound, drug Puromycin Sigma- Aldrich Cat#:P7255 Chemical compound, drug Paclitaxel Cayman Chem Cat#:10461 Chemical compound, drug PARP inhibitor PJ34 Enzo Cat#:ALX- 270–289 Software, algorithm Mascot v2.7.0 Matrix Science RRID:SCR_014322 Software, algorithm Scaffold v4.9.0 Proteome Software RRID:SCR_014321 Software, algorithm ImageJ FIJI NIH RRID:SCR_002285 Software, algorithm BioInfoRx https://bioinforx.com/ apps/venn.php Software, algorithm Nematode genome comparison browser http://nemates.org/MA/ progs/overlap_stats.html Software, algorithm ShinyGO v0.67 & v0.77 Ge et al., 2020 Software, algorithm Metascape v3.5 Zhou et al., 2019 Software, algorithm Panther v17.0 Thomas et al., 2022 Software, algorithm Revigo Supek et al., 2011 Software, algorithm STRING v11.5 Szklarczyk et al., 2023 Software, algorithm Cytoscape Doncheva et al., 2023 Software, algorithm R2 http://r2.amc.nl Software, algorithm dcGO Enrichment mining service Fang and Gough, 2013 Software, algorithm STAR v2.7.7.a Dobin et al., 2013 Software, algorithm rMATS v4.1.2 Shen et al., 2014 Software, algorithm rmats2sashimiplot tool https://github.com/ Xinglab/rmats2sashimiplot; Xinglab, 2024 Continued Continued on next page Breunig, Lei, Montalbano et al. eLife 2024;13:RP98152.

    Techniques: Immunofluorescence, Control, Staining, Fluorescence, Software