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Bethyl anti pelp1 polyclonal antibody

( A ) Cryo-EM structure of the human pre-60S ribosome with the rixosome bound [PDB ID:8FL2 ]. Only the conserved scaffolding core of the rixosome <t>(PELP1</t> Rix1 domain, WDR18, and TEX10) is visible in the structure. Also see fig. S1. The rixosome core is formed by two copies of the PELP1 Rix1 domain (shades of teal), two copies of WDR18 (shades of orange), and one copy of TEX10 (pink). ( B ) Structure of the rixosome core indicating the putative positions of the PELP1 C-terminal IDRs. It remains unclear how the three rixosome enzyme LAS1L (ribonuclease, RNase), NOL9 (poly-nucleotide kinase, PNK), and SENP3 (SUMO protease) associate with the rixosome core. ( C ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 6) used to affinity purify the rixosome in the co-immunoprecipitation (co-IP) experiment displayed in (D). Checkmarks and X marks denote qualitative binding results for specific rixosome components to the PELP1 variant used for reconstitution and co-IP. * denotes a notable decrease (but still detectable) in signal for specific rixosome components. ( D ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins qualitatively identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP from human cells of PELP1 variants in (C). EV denotes empty vector control.

Anti Pelp1 Polyclonal Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti pelp1 polyclonal antibody - by Bioz Stars, 2026-03

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1) Product Images from "PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex"

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

Journal: Science Advances

doi: 10.1126/sciadv.adw4603

( A ) Cryo-EM structure of the human pre-60S ribosome with the rixosome bound [PDB ID:8FL2 ]. Only the conserved scaffolding core of the rixosome (PELP1 Rix1 domain, WDR18, and TEX10) is visible in the structure. Also see fig. S1. The rixosome core is formed by two copies of the PELP1 Rix1 domain (shades of teal), two copies of WDR18 (shades of orange), and one copy of TEX10 (pink). ( B ) Structure of the rixosome core indicating the putative positions of the PELP1 C-terminal IDRs. It remains unclear how the three rixosome enzyme LAS1L (ribonuclease, RNase), NOL9 (poly-nucleotide kinase, PNK), and SENP3 (SUMO protease) associate with the rixosome core. ( C ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 6) used to affinity purify the rixosome in the co-immunoprecipitation (co-IP) experiment displayed in (D). Checkmarks and X marks denote qualitative binding results for specific rixosome components to the PELP1 variant used for reconstitution and co-IP. * denotes a notable decrease (but still detectable) in signal for specific rixosome components. ( D ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins qualitatively identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP from human cells of PELP1 variants in (C). EV denotes empty vector control.

Figure Legend Snippet: ( A ) Cryo-EM structure of the human pre-60S ribosome with the rixosome bound [PDB ID:8FL2 ]. Only the conserved scaffolding core of the rixosome (PELP1 Rix1 domain, WDR18, and TEX10) is visible in the structure. Also see fig. S1. The rixosome core is formed by two copies of the PELP1 Rix1 domain (shades of teal), two copies of WDR18 (shades of orange), and one copy of TEX10 (pink). ( B ) Structure of the rixosome core indicating the putative positions of the PELP1 C-terminal IDRs. It remains unclear how the three rixosome enzyme LAS1L (ribonuclease, RNase), NOL9 (poly-nucleotide kinase, PNK), and SENP3 (SUMO protease) associate with the rixosome core. ( C ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 6) used to affinity purify the rixosome in the co-immunoprecipitation (co-IP) experiment displayed in (D). Checkmarks and X marks denote qualitative binding results for specific rixosome components to the PELP1 variant used for reconstitution and co-IP. * denotes a notable decrease (but still detectable) in signal for specific rixosome components. ( D ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins qualitatively identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP from human cells of PELP1 variants in (C). EV denotes empty vector control.

Techniques Used: Cryo-EM Sample Prep, Scaffolding, Variant Assay, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Plasmid Preparation, Control

( A ) Human rixosome cartoon indicating both copies of PELP1’s C-terminal IDR, with specific areas of interest labeled. ( B ) Sequence and structural characteristics of the PELP1 C-terminal IDR (residues 642 to 1130). AlphaFold3 secondary (II) structure predictions, disorder propensity (%), and polyproline secondary structure propensity (PPII score) are displayed per residue of the PELP1 IDR. ( C ) Amino acid distribution plots for negative [D (red) and E (yellow)], positive [R (brown) and K (blue)], glycine (G, gray), and proline (P, green) residues. ( D ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 7) used in the co-IP experiment displayed in (E) and (F). Checkmarks and X marks denote qualitative binding results for SENP3, MDN1, or histones to the PELP1 variant used for reconstitution and co-IP. ( E ) SDS-PAGE and Western blot using antibodies for the exogenous rixosome proteins upon co-IP from human cells of PELP1 variants in (D) + or − a nonspecific nuclease in the lysis buffer. These isolated rixosome complexes were natively eluted off the anti-FLAG affinity gel and analyzed by SDS-PAGE and total protein staining to qualitatively identify endogenous interactors copurifying with the human rixosome. Red arrows at bottom denote sample lanes with histone copurification. ( F ) SDS-PAGE and Western blot using specific antibodies for the endogenous protein targets copurifying with PELP1/rixosome complexes upon co-IP from human cells in (E) + a nonspecific nuclease in the lysis buffer. MDN1 qualitative binding was confirmed by Western blot. Nucleosome core histones (H2A-H2B-H3-H4) qualitative binding was determined to be specific to the PELP1 GAR and confirmed by Western blot to H3. ( G ) SDS-PAGE and Western blot from an endogenous PELP1 co-IP from HEK293FT cells + a nonspecific nuclease in the lysis buffer using PELP1-specific antibody. Endogenous MDN1 and H3 were detected by Western blot.

Figure Legend Snippet: ( A ) Human rixosome cartoon indicating both copies of PELP1’s C-terminal IDR, with specific areas of interest labeled. ( B ) Sequence and structural characteristics of the PELP1 C-terminal IDR (residues 642 to 1130). AlphaFold3 secondary (II) structure predictions, disorder propensity (%), and polyproline secondary structure propensity (PPII score) are displayed per residue of the PELP1 IDR. ( C ) Amino acid distribution plots for negative [D (red) and E (yellow)], positive [R (brown) and K (blue)], glycine (G, gray), and proline (P, green) residues. ( D ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 7) used in the co-IP experiment displayed in (E) and (F). Checkmarks and X marks denote qualitative binding results for SENP3, MDN1, or histones to the PELP1 variant used for reconstitution and co-IP. ( E ) SDS-PAGE and Western blot using antibodies for the exogenous rixosome proteins upon co-IP from human cells of PELP1 variants in (D) + or − a nonspecific nuclease in the lysis buffer. These isolated rixosome complexes were natively eluted off the anti-FLAG affinity gel and analyzed by SDS-PAGE and total protein staining to qualitatively identify endogenous interactors copurifying with the human rixosome. Red arrows at bottom denote sample lanes with histone copurification. ( F ) SDS-PAGE and Western blot using specific antibodies for the endogenous protein targets copurifying with PELP1/rixosome complexes upon co-IP from human cells in (E) + a nonspecific nuclease in the lysis buffer. MDN1 qualitative binding was confirmed by Western blot. Nucleosome core histones (H2A-H2B-H3-H4) qualitative binding was determined to be specific to the PELP1 GAR and confirmed by Western blot to H3. ( G ) SDS-PAGE and Western blot from an endogenous PELP1 co-IP from HEK293FT cells + a nonspecific nuclease in the lysis buffer using PELP1-specific antibody. Endogenous MDN1 and H3 were detected by Western blot.

Techniques Used: Labeling, Sequencing, Residue, Variant Assay, Construct, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Lysis, Isolation, Staining, Copurification

( A ) Cartoon of the human rixosome highlighting the specific PELP1 IDR-MDN1 interaction region of interest. ( B ) Schematic of human AAA-ATPase MDN1 noting major protein domains. The six N-terminal AAA-ATPase domains are specifically noted as D1-D6. Also see fig. S3. ( C ) AlphaFold3 structural prediction of the entire human MDN1 AAA-ATPase domain bound to the experimentally determined region of the PELP1 IDR. Exact protein residues used for the prediction are noted in the panel. AlphaFold3 predicts a small helix of PELP1 (MDN1 interacting helix, MIH) binds to the D2H2α insert of MDN1. Also see fig. S4. ( D ) Zoom from (C) of predicted MDN1-PELP1 binding interface with PELP1 F1112 forming an aromatic anchor into a pocket of the MDN1 D2H2a. Also see fig. S3. ( E ) Multiple sequence alignment of PELP1 MIH region responsible for binding MDN1 D2H2a. Species abbreviations: Homo sapiens (H.s.), Mus musculus (M.m.), Bos taurus (B.t.), Aquila chrysaetos (A.c.), Xenopus laevis (X.l.), Danio rerio (D.r.), Branchiostoma lanceolatum (B.l.), Acanthaster planci (A.p.), Mytilus coruscus (M.c.), Drosophila melanogaster (D.m.), Dictyostelium discoideum (D.d.), Schizosaccharomyces pombe (S.p.), Saccharomyces cerevisiae (S.c.). ( F ) Schematic of C-terminal FLAG tagged PELP1 truncations, deletions, and point mutants (1 to 6) used for co-IPs to assay endogenous MDN1 binding from human cells in (G) and (H). ( G ) SDS-PAGE and Western blot using antibodies for the endogenous MDN1 protein and exogenous rixosome components upon co-IP of PELP1 variants in (F). All PELP1 MIH mutants failed to bind qualitatively to MDN1, supporting the predicted structural model of PELP1 MIH binding to MDN1 D2H2a in (C). ( H ) SDS-PAGE, Western blot, and total protein stain of FLAG PELP1 C terminus (residues 967 to 1130) alone copurifying MDN1 from cells. ( I ) A proposed mechanism for MDN1 regulation by PELP1/the rixosome in large ribosomal subunit maturation.

Figure Legend Snippet: ( A ) Cartoon of the human rixosome highlighting the specific PELP1 IDR-MDN1 interaction region of interest. ( B ) Schematic of human AAA-ATPase MDN1 noting major protein domains. The six N-terminal AAA-ATPase domains are specifically noted as D1-D6. Also see fig. S3. ( C ) AlphaFold3 structural prediction of the entire human MDN1 AAA-ATPase domain bound to the experimentally determined region of the PELP1 IDR. Exact protein residues used for the prediction are noted in the panel. AlphaFold3 predicts a small helix of PELP1 (MDN1 interacting helix, MIH) binds to the D2H2α insert of MDN1. Also see fig. S4. ( D ) Zoom from (C) of predicted MDN1-PELP1 binding interface with PELP1 F1112 forming an aromatic anchor into a pocket of the MDN1 D2H2a. Also see fig. S3. ( E ) Multiple sequence alignment of PELP1 MIH region responsible for binding MDN1 D2H2a. Species abbreviations: Homo sapiens (H.s.), Mus musculus (M.m.), Bos taurus (B.t.), Aquila chrysaetos (A.c.), Xenopus laevis (X.l.), Danio rerio (D.r.), Branchiostoma lanceolatum (B.l.), Acanthaster planci (A.p.), Mytilus coruscus (M.c.), Drosophila melanogaster (D.m.), Dictyostelium discoideum (D.d.), Schizosaccharomyces pombe (S.p.), Saccharomyces cerevisiae (S.c.). ( F ) Schematic of C-terminal FLAG tagged PELP1 truncations, deletions, and point mutants (1 to 6) used for co-IPs to assay endogenous MDN1 binding from human cells in (G) and (H). ( G ) SDS-PAGE and Western blot using antibodies for the endogenous MDN1 protein and exogenous rixosome components upon co-IP of PELP1 variants in (F). All PELP1 MIH mutants failed to bind qualitatively to MDN1, supporting the predicted structural model of PELP1 MIH binding to MDN1 D2H2a in (C). ( H ) SDS-PAGE, Western blot, and total protein stain of FLAG PELP1 C terminus (residues 967 to 1130) alone copurifying MDN1 from cells. ( I ) A proposed mechanism for MDN1 regulation by PELP1/the rixosome in large ribosomal subunit maturation.

Techniques Used: Structural Proteomics, Binding Assay, Sequencing, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Staining

$( A ) Cartoon of the human rixosome highlighting the GAR region. ( B ) Cartoon of the in vitro histone chaperoning assay illustrating potential IDR histone interactions that could form upon incubating PELP1 with H2A-H2B dimers and H3-H4 tetramers. Created in BioRender. Gordon, J. (2025) https://BioRender.com/x62f662 . ( C ) Multiple sequence alignment of PELP1 GAR. Highly conserved stretches of glutamic acid residues are highlighted in magenta. Species abbreviations are the same as . ( D to F ) Histone chaperone assay with PELP1 IDR region 800 to 1130aa (D), PELP1 IDR region 887 to 966aa (E), and PELP1 IDR region 967 to 1140aa (negative control) (F). Postdialysis histone octamer and MBP-PELP1 variant mixed samples were subject to S200 gel filtration chromatography to assess histone binding to PELP1 GAR. Mixed samples at 1:1 (blue curve) and 2:1 (red curve) ratios exhibited migration shifts compared to unmixed controls, indicating stable binding. SDS-PAGE of peak fractions also indicate the presence of bound complexes. Histone octamer alone control sample exhibits two peaks corresponding to dissociated histone H2A-H2B dimers and H3-H4 tetramers. ( G ) Structure of the nucleosome core particle [PDB ID: 3LZ0 ]. DNA is colored on the basis of electrostatic surface. ( H ) AlphaFold3 structural prediction of the human histone octamer bound to one copy of the PELP1 GAR, 1:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel (full-length human H2A 2 -H2B 2 -H3 2 -H4 2 used for octamer prediction). PELP1 GAR is predicted to be helical when bound to histones and is depicted as electrostatic surface representation. Also see fig. S5. ( I ) AlphaFold3 structural prediction of the human histone octamer bound to two copies of the PELP1 GAR, 2:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel and same as (H). Also see fig. S6.$
Figure Legend Snippet: ( A ) Cartoon of the human rixosome highlighting the GAR region. ( B ) Cartoon of the in vitro histone chaperoning assay illustrating potential IDR histone interactions that could form upon incubating PELP1 with H2A-H2B dimers and H3-H4 tetramers. Created in BioRender. Gordon, J. (2025) https://BioRender.com/x62f662 . ( C ) Multiple sequence alignment of PELP1 GAR. Highly conserved stretches of glutamic acid residues are highlighted in magenta. Species abbreviations are the same as . ( D to F ) Histone chaperone assay with PELP1 IDR region 800 to 1130aa (D), PELP1 IDR region 887 to 966aa (E), and PELP1 IDR region 967 to 1140aa (negative control) (F). Postdialysis histone octamer and MBP-PELP1 variant mixed samples were subject to S200 gel filtration chromatography to assess histone binding to PELP1 GAR. Mixed samples at 1:1 (blue curve) and 2:1 (red curve) ratios exhibited migration shifts compared to unmixed controls, indicating stable binding. SDS-PAGE of peak fractions also indicate the presence of bound complexes. Histone octamer alone control sample exhibits two peaks corresponding to dissociated histone H2A-H2B dimers and H3-H4 tetramers. ( G ) Structure of the nucleosome core particle [PDB ID: 3LZ0 ]. DNA is colored on the basis of electrostatic surface. ( H ) AlphaFold3 structural prediction of the human histone octamer bound to one copy of the PELP1 GAR, 1:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel (full-length human H2A 2 -H2B 2 -H3 2 -H4 2 used for octamer prediction). PELP1 GAR is predicted to be helical when bound to histones and is depicted as electrostatic surface representation. Also see fig. S5. ( I ) AlphaFold3 structural prediction of the human histone octamer bound to two copies of the PELP1 GAR, 2:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel and same as (H). Also see fig. S6.

Techniques Used: In Vitro, Sequencing, Negative Control, Variant Assay, Filtration, Chromatography, Binding Assay, Migration, SDS Page, Control, Structural Proteomics

( A ) Cartoon of the human rixosome highlighting the SENP3-interacting region. ( B ) N-terminal FLAG-tagged SENP3 truncations (1 to 3) used in the co-IP experiment displayed in (C). Checkmarks and X marks denote qualitative binding results for specific rixosome components in (C) to the SENP3 truncation used for co-IP. ( C ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP with SENP3 truncations. ( D ) X-ray crystal structure of the SENP3 protease domain (311 to 574aa, dark blue) C532S mutant bound to the PELP1 SLiM peptide (764 to 781aa, teal) at 2.93 Å resolution. SENP3 active site with catalytic triad residues is circled in red. ( E ) PELP1 SLiM residues 765 to 767 (FVH motif) and 776 to 778 (VEI motif) form antiparallel β-strand interactions with SENP3 at β6* and β7, respectively. ( F ) The PELP1 SLiM binding interface with the SENP3 protease domain surface. ( G ) The PELP1 FVH motif secondary structure backbone interactions with SENP3 β6*. Putative backbone hydrogen bonding interactions are illustrated by dashed lines. Experimental electron density is displayed as a composite omit map contoured at σ = 0.152 e/Å 3 . ( H ) The PELP1 VEI motif secondary structure backbone interactions with SENP3 β7. Putative backbone hydrogen bonding interactions are illustrated by dashed lines with the experimental density shown as in (G). ( I ) Multiple sequence alignment of PELP1 SLiM and flanking regions. Species abbreviations are the same as . ( J ) Schematic of C-terminal FLAG-tagged PELP1 mutant constructs (1 to 5) used in the co-IP experiment displayed in (K). Checkmarks and X marks denote qualitative binding results for SENP3 (K) to the PELP1 mutant. ( K ) SDS-PAGE and Western blot identified the presence or absence of SENP3 and other rixosome proteins upon co-IP from human cells of PELP1 mutant constructs in (J).

Figure Legend Snippet: ( A ) Cartoon of the human rixosome highlighting the SENP3-interacting region. ( B ) N-terminal FLAG-tagged SENP3 truncations (1 to 3) used in the co-IP experiment displayed in (C). Checkmarks and X marks denote qualitative binding results for specific rixosome components in (C) to the SENP3 truncation used for co-IP. ( C ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP with SENP3 truncations. ( D ) X-ray crystal structure of the SENP3 protease domain (311 to 574aa, dark blue) C532S mutant bound to the PELP1 SLiM peptide (764 to 781aa, teal) at 2.93 Å resolution. SENP3 active site with catalytic triad residues is circled in red. ( E ) PELP1 SLiM residues 765 to 767 (FVH motif) and 776 to 778 (VEI motif) form antiparallel β-strand interactions with SENP3 at β6* and β7, respectively. ( F ) The PELP1 SLiM binding interface with the SENP3 protease domain surface. ( G ) The PELP1 FVH motif secondary structure backbone interactions with SENP3 β6*. Putative backbone hydrogen bonding interactions are illustrated by dashed lines. Experimental electron density is displayed as a composite omit map contoured at σ = 0.152 e/Å 3 . ( H ) The PELP1 VEI motif secondary structure backbone interactions with SENP3 β7. Putative backbone hydrogen bonding interactions are illustrated by dashed lines with the experimental density shown as in (G). ( I ) Multiple sequence alignment of PELP1 SLiM and flanking regions. Species abbreviations are the same as . ( J ) Schematic of C-terminal FLAG-tagged PELP1 mutant constructs (1 to 5) used in the co-IP experiment displayed in (K). Checkmarks and X marks denote qualitative binding results for SENP3 (K) to the PELP1 mutant. ( K ) SDS-PAGE and Western blot identified the presence or absence of SENP3 and other rixosome proteins upon co-IP from human cells of PELP1 mutant constructs in (J).

Techniques Used: Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Mutagenesis, Sequencing, Construct

$( A ) Size exclusion chromatography (SEC) curves exhibiting the formation of a complex between SENP3 protease domain 302 to 574aa and PELP1 SLiM -containing peptide 761 to 796aa. SDS-PAGE and total protein staining of SEC fractions are displayed below the x axis. ( B ) AlphaFold3 structural models of proSUMO substrates used in endopeptidase cleavage assays. Sequences of the proSUMO C-terminal tails (cleavage sites labeled with arrows) are shown below the structural models. ( C ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM endopeptidase activity assays against proSUMO1, −2, and −3 substrates. Decreasing concentrations (2500 to 0.12 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 5 μM proSUMO substrate. Cleaved proSUMO product is labeled with asterisks. Quantification curves representing percent (%) proSUMO endopeptidase cleavage correspond to the gel images and the enzyme concentration range was 1000 to 15.63 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and total protein stain of in vitro SENP3 (C532A) ± MBP-PELP1 SLiM endopeptidase activity assay, illustrating no activity. ( E ) Quantification curves representing percent (%) proSUMO2 endopeptidase cleavage by SENP protease domain + PELP1 SLiM , SENP3 protease domain alone, or SENP5 protease domain alone during a time course (0 to 450 s). Enzyme concentration was kept constant at 1000 nM along with substrate concentration at 5 μM. Percent cleavage was calculated as in (C). SD was calculated from three independent assay samples ( n = 3). Raw gel images are displayed in fig. S13A. ( F ) Differential scanning fluorimetry curves exhibiting the thermal stabilization of the SENP3 protease domain upon addition of short (“s,” amino acids 764 to 781) and long (“l,” amino acids 764 to 792) PELP1 SLIM peptides. Boltzmann and first derivative curves are shown on the top and bottom of the panel, respectively. Values for change in T m is only shown for the first derivative.$
Figure Legend Snippet: ( A ) Size exclusion chromatography (SEC) curves exhibiting the formation of a complex between SENP3 protease domain 302 to 574aa and PELP1 SLiM -containing peptide 761 to 796aa. SDS-PAGE and total protein staining of SEC fractions are displayed below the x axis. ( B ) AlphaFold3 structural models of proSUMO substrates used in endopeptidase cleavage assays. Sequences of the proSUMO C-terminal tails (cleavage sites labeled with arrows) are shown below the structural models. ( C ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM endopeptidase activity assays against proSUMO1, −2, and −3 substrates. Decreasing concentrations (2500 to 0.12 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 5 μM proSUMO substrate. Cleaved proSUMO product is labeled with asterisks. Quantification curves representing percent (%) proSUMO endopeptidase cleavage correspond to the gel images and the enzyme concentration range was 1000 to 15.63 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and total protein stain of in vitro SENP3 (C532A) ± MBP-PELP1 SLiM endopeptidase activity assay, illustrating no activity. ( E ) Quantification curves representing percent (%) proSUMO2 endopeptidase cleavage by SENP protease domain + PELP1 SLiM , SENP3 protease domain alone, or SENP5 protease domain alone during a time course (0 to 450 s). Enzyme concentration was kept constant at 1000 nM along with substrate concentration at 5 μM. Percent cleavage was calculated as in (C). SD was calculated from three independent assay samples ( n = 3). Raw gel images are displayed in fig. S13A. ( F ) Differential scanning fluorimetry curves exhibiting the thermal stabilization of the SENP3 protease domain upon addition of short (“s,” amino acids 764 to 781) and long (“l,” amino acids 764 to 792) PELP1 SLIM peptides. Boltzmann and first derivative curves are shown on the top and bottom of the panel, respectively. Values for change in T m is only shown for the first derivative.

Techniques Used: Size-exclusion Chromatography, SDS Page, Staining, Labeling, In Vitro, Activity Assay, Incubation, Concentration Assay

( A ) AlphaFold3 structural model of MBP-Flag-NPM1 (240 to 294aa) SUMO2 conjugate, with the cleavage site indicated with a red arrow. NPM1 SUMOylation site K263 depicted. ( B ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM isopeptidase activity assays against MBP-Flag-NPM1-SUMO2 conjugates. Decreasing concentrations (1000 to 3.9 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 15 μl of on-resin NPM1-SUMO2 conjugates. Cleaved free SUMO product is labeled. Red * on bands in control lanes denote MBP-Flag-NPM1 that copurifies without SUMO2 conjugation. ( C ) Quantification curves representing percent (%) cleavage of substrate over time correspond to the gel images in fig. S13B and the enzyme concentration used was 500 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and Western blot of HA-tagged SUMO2 conjugated proteins detected in HEK293FT cell lysate. Changes in amount of HA-SUMO2 conjugated proteins in vivo was assayed after transfecting cells with wild-type or C532A catalytic mutant SENP3 in combination with titrated transfection of wild-type PELP1. Decreasing amounts of transfected DNA expressing exogenous PELP1 with wild-type SENP3 resulted in an overall dose-dependent increase of HA-SUMO2–conjugated proteins similar to that observed with SENP3 C532A (catalytic-dead mutant). ( E ) Same as (D) except HA-tagged SUMO1 was used.

Figure Legend Snippet: ( A ) AlphaFold3 structural model of MBP-Flag-NPM1 (240 to 294aa) SUMO2 conjugate, with the cleavage site indicated with a red arrow. NPM1 SUMOylation site K263 depicted. ( B ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM isopeptidase activity assays against MBP-Flag-NPM1-SUMO2 conjugates. Decreasing concentrations (1000 to 3.9 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 15 μl of on-resin NPM1-SUMO2 conjugates. Cleaved free SUMO product is labeled. Red * on bands in control lanes denote MBP-Flag-NPM1 that copurifies without SUMO2 conjugation. ( C ) Quantification curves representing percent (%) cleavage of substrate over time correspond to the gel images in fig. S13B and the enzyme concentration used was 500 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and Western blot of HA-tagged SUMO2 conjugated proteins detected in HEK293FT cell lysate. Changes in amount of HA-SUMO2 conjugated proteins in vivo was assayed after transfecting cells with wild-type or C532A catalytic mutant SENP3 in combination with titrated transfection of wild-type PELP1. Decreasing amounts of transfected DNA expressing exogenous PELP1 with wild-type SENP3 resulted in an overall dose-dependent increase of HA-SUMO2–conjugated proteins similar to that observed with SENP3 C532A (catalytic-dead mutant). ( E ) Same as (D) except HA-tagged SUMO1 was used.

Techniques Used: SDS Page, Staining, In Vitro, Activity Assay, Incubation, Labeling, Control, Conjugation Assay, Concentration Assay, Western Blot, In Vivo, Mutagenesis, Transfection, Expressing

( A ) Swiss army knife model of the rixosome. The PELP1-WDR18-TEX10 scaffolding core is analogous to the housing unit of the knife from which the different diverse tools extend outward, including scissors (RNase PNK RNA cutting), saw blade (SENP3 protease), and corkscrew (MDN1 ATPase motor remodeling). ( B ) Cartoon structural model of the full human rixosome. The scaffolding core (PELP1-WDR18-TEX10) represents the stable core of the rixosome complex from which enzymatic components are connected. The RNA processing complex RNase PNK (LAS1L-NOL9) is physically and flexibly tethered to the stable scaffolding core. The deSUMOylation enzyme SENP3 and AAA-ATPase MDN1 are distinctly integrated with the rixosome through PELP1’s C-terminal IDRs. ( C ) Proposed architectural model of the human rixosome bound to the pre-60S subunit [PDB ID: 8FL2 ]. PELP1’s IDRs organize the enzymatic functions of SENP3 and MDN1. ( D ) Proposed architectural model of the human rixosome bound to polycomb and functioning in gene silencing at facultative heterochromatin. PELP1’s IDRs likely organize multiple enzymatic and nonenzymatic functions at heterochromatin, including SENP3 and MDN1 activity, and putative histone chaperoning by the GAR.

Figure Legend Snippet: ( A ) Swiss army knife model of the rixosome. The PELP1-WDR18-TEX10 scaffolding core is analogous to the housing unit of the knife from which the different diverse tools extend outward, including scissors (RNase PNK RNA cutting), saw blade (SENP3 protease), and corkscrew (MDN1 ATPase motor remodeling). ( B ) Cartoon structural model of the full human rixosome. The scaffolding core (PELP1-WDR18-TEX10) represents the stable core of the rixosome complex from which enzymatic components are connected. The RNA processing complex RNase PNK (LAS1L-NOL9) is physically and flexibly tethered to the stable scaffolding core. The deSUMOylation enzyme SENP3 and AAA-ATPase MDN1 are distinctly integrated with the rixosome through PELP1’s C-terminal IDRs. ( C ) Proposed architectural model of the human rixosome bound to the pre-60S subunit [PDB ID: 8FL2 ]. PELP1’s IDRs organize the enzymatic functions of SENP3 and MDN1. ( D ) Proposed architectural model of the human rixosome bound to polycomb and functioning in gene silencing at facultative heterochromatin. PELP1’s IDRs likely organize multiple enzymatic and nonenzymatic functions at heterochromatin, including SENP3 and MDN1 activity, and putative histone chaperoning by the GAR.

Techniques Used: Scaffolding, Activity Assay

Image Search Results

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Cryo-EM structure of the human pre-60S ribosome with the rixosome bound [PDB ID:8FL2 ]. Only the conserved scaffolding core of the rixosome (PELP1 Rix1 domain, WDR18, and TEX10) is visible in the structure. Also see fig. S1. The rixosome core is formed by two copies of the PELP1 Rix1 domain (shades of teal), two copies of WDR18 (shades of orange), and one copy of TEX10 (pink). ( B ) Structure of the rixosome core indicating the putative positions of the PELP1 C-terminal IDRs. It remains unclear how the three rixosome enzyme LAS1L (ribonuclease, RNase), NOL9 (poly-nucleotide kinase, PNK), and SENP3 (SUMO protease) associate with the rixosome core. ( C ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 6) used to affinity purify the rixosome in the co-immunoprecipitation (co-IP) experiment displayed in (D). Checkmarks and X marks denote qualitative binding results for specific rixosome components to the PELP1 variant used for reconstitution and co-IP. * denotes a notable decrease (but still detectable) in signal for specific rixosome components. ( D ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins qualitatively identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP from human cells of PELP1 variants in (C). EV denotes empty vector control.

Article Snippet: Antibodies used for Western blotting in this study include the following: Polyclonal anti-FLAG produced in rabbit (Sigma-Aldrich, Cat# F7425, RRID:AB_439687), Monoclonal anti-FLAG M2-peroxidase HRP conjugate produced in mouse (Sigma-Aldrich, Cat# A8592, RRID:AB_439702), Monoclonal anti-HA produced in mouse (Thermo Fisher Scientific, Cat# 26183, RRID:AB_10978021), Monoclonal anti-GFP produced in mouse (Sigma-Aldrich, Cat# 11814460001, RRID:AB_390913), Monoclonal anti-MYC (clone 4A6) produced in mouse (Sigma-Aldrich, Cat# 05-724, RRID:AB_11211891), anti-PELP1 polyclonal antibody produced in rabbit (Bethyl, Cat# A300-180A, RRID:AB_242526), anti-WDR18 polyclonal antibody produced in rabbit (Sigma-Aldrich, Cat# HPA050200, RRID:AB_2681049), anti-TEX10 polyclonal antibody produced in rabbit (Thermo Fisher Scientific, Cat# 720257, RRID:AB_2633219), anti-SENP3 polyclonal antibody produced in rabbit (Bethyl, Cat# A303-139A, RRID:AB_10895725), anti-LAS1L polyclonal antibody produced in rabbit (Proteintech, Cat# 16010-1-AP, RRID:AB_2132810), anti-NOL9 polyclonal antibody produced in rabbit (Abcam, Cat# ab103207, RRID:AB_10712240), anti-MDN1 polyclonal antibody produced in rabbit (Sigma-Aldrich, Cat# HPA029666, RRID:AB_10600888), anti-Histone H3 polyclonal antibody produced in rabbit (Sigma-Aldrich, Cat# H0164, RRID:AB_532248), anti-RING1B monoclonal antibody produced in rabbit (Cell Signaling Technology, Cat# 5694, RRID:AB_10705604), anti-BMI1 polyclonal antibody produced in rabbit (Proteintech, Cat# 10832-1-AP, RRID:AB_2065392), anti-RYBP polyclonal antibody produced in rabbit (Proteintech, Cat# 11365-1-AP, RRID:AB_2285461), anti-mouse IgG (whole molecule)-Peroxidase antibody produced in rabbit (Sigma-Aldrich, Cat# A9044, RRID:AB_258431), anti-rabbit IgG, HRP conjugate antibody produced in goat (Sigma-Aldrich, Cat# 12-348, RRID:AB_390191), Normal Rabbit IgG (Millipore, Cat# 12-370, RRID:AB_145841).

Techniques: Cryo-EM Sample Prep, Scaffolding, Variant Assay, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Plasmid Preparation, Control

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Human rixosome cartoon indicating both copies of PELP1’s C-terminal IDR, with specific areas of interest labeled. ( B ) Sequence and structural characteristics of the PELP1 C-terminal IDR (residues 642 to 1130). AlphaFold3 secondary (II) structure predictions, disorder propensity (%), and polyproline secondary structure propensity (PPII score) are displayed per residue of the PELP1 IDR. ( C ) Amino acid distribution plots for negative [D (red) and E (yellow)], positive [R (brown) and K (blue)], glycine (G, gray), and proline (P, green) residues. ( D ) Schematic of C-terminal FLAG-tagged PELP1 variant/truncation constructs (1 to 7) used in the co-IP experiment displayed in (E) and (F). Checkmarks and X marks denote qualitative binding results for SENP3, MDN1, or histones to the PELP1 variant used for reconstitution and co-IP. ( E ) SDS-PAGE and Western blot using antibodies for the exogenous rixosome proteins upon co-IP from human cells of PELP1 variants in (D) + or − a nonspecific nuclease in the lysis buffer. These isolated rixosome complexes were natively eluted off the anti-FLAG affinity gel and analyzed by SDS-PAGE and total protein staining to qualitatively identify endogenous interactors copurifying with the human rixosome. Red arrows at bottom denote sample lanes with histone copurification. ( F ) SDS-PAGE and Western blot using specific antibodies for the endogenous protein targets copurifying with PELP1/rixosome complexes upon co-IP from human cells in (E) + a nonspecific nuclease in the lysis buffer. MDN1 qualitative binding was confirmed by Western blot. Nucleosome core histones (H2A-H2B-H3-H4) qualitative binding was determined to be specific to the PELP1 GAR and confirmed by Western blot to H3. ( G ) SDS-PAGE and Western blot from an endogenous PELP1 co-IP from HEK293FT cells + a nonspecific nuclease in the lysis buffer using PELP1-specific antibody. Endogenous MDN1 and H3 were detected by Western blot.

Techniques: Labeling, Sequencing, Residue, Variant Assay, Construct, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Lysis, Isolation, Staining, Copurification

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Cartoon of the human rixosome highlighting the specific PELP1 IDR-MDN1 interaction region of interest. ( B ) Schematic of human AAA-ATPase MDN1 noting major protein domains. The six N-terminal AAA-ATPase domains are specifically noted as D1-D6. Also see fig. S3. ( C ) AlphaFold3 structural prediction of the entire human MDN1 AAA-ATPase domain bound to the experimentally determined region of the PELP1 IDR. Exact protein residues used for the prediction are noted in the panel. AlphaFold3 predicts a small helix of PELP1 (MDN1 interacting helix, MIH) binds to the D2H2α insert of MDN1. Also see fig. S4. ( D ) Zoom from (C) of predicted MDN1-PELP1 binding interface with PELP1 F1112 forming an aromatic anchor into a pocket of the MDN1 D2H2a. Also see fig. S3. ( E ) Multiple sequence alignment of PELP1 MIH region responsible for binding MDN1 D2H2a. Species abbreviations: Homo sapiens (H.s.), Mus musculus (M.m.), Bos taurus (B.t.), Aquila chrysaetos (A.c.), Xenopus laevis (X.l.), Danio rerio (D.r.), Branchiostoma lanceolatum (B.l.), Acanthaster planci (A.p.), Mytilus coruscus (M.c.), Drosophila melanogaster (D.m.), Dictyostelium discoideum (D.d.), Schizosaccharomyces pombe (S.p.), Saccharomyces cerevisiae (S.c.). ( F ) Schematic of C-terminal FLAG tagged PELP1 truncations, deletions, and point mutants (1 to 6) used for co-IPs to assay endogenous MDN1 binding from human cells in (G) and (H). ( G ) SDS-PAGE and Western blot using antibodies for the endogenous MDN1 protein and exogenous rixosome components upon co-IP of PELP1 variants in (F). All PELP1 MIH mutants failed to bind qualitatively to MDN1, supporting the predicted structural model of PELP1 MIH binding to MDN1 D2H2a in (C). ( H ) SDS-PAGE, Western blot, and total protein stain of FLAG PELP1 C terminus (residues 967 to 1130) alone copurifying MDN1 from cells. ( I ) A proposed mechanism for MDN1 regulation by PELP1/the rixosome in large ribosomal subunit maturation.

Techniques: Structural Proteomics, Binding Assay, Sequencing, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Staining

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Cartoon of the human rixosome highlighting the GAR region. ( B ) Cartoon of the in vitro histone chaperoning assay illustrating potential IDR histone interactions that could form upon incubating PELP1 with H2A-H2B dimers and H3-H4 tetramers. Created in BioRender. Gordon, J. (2025) https://BioRender.com/x62f662 . ( C ) Multiple sequence alignment of PELP1 GAR. Highly conserved stretches of glutamic acid residues are highlighted in magenta. Species abbreviations are the same as . ( D to F ) Histone chaperone assay with PELP1 IDR region 800 to 1130aa (D), PELP1 IDR region 887 to 966aa (E), and PELP1 IDR region 967 to 1140aa (negative control) (F). Postdialysis histone octamer and MBP-PELP1 variant mixed samples were subject to S200 gel filtration chromatography to assess histone binding to PELP1 GAR. Mixed samples at 1:1 (blue curve) and 2:1 (red curve) ratios exhibited migration shifts compared to unmixed controls, indicating stable binding. SDS-PAGE of peak fractions also indicate the presence of bound complexes. Histone octamer alone control sample exhibits two peaks corresponding to dissociated histone H2A-H2B dimers and H3-H4 tetramers. ( G ) Structure of the nucleosome core particle [PDB ID: 3LZ0 ]. DNA is colored on the basis of electrostatic surface. ( H ) AlphaFold3 structural prediction of the human histone octamer bound to one copy of the PELP1 GAR, 1:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel (full-length human H2A 2 -H2B 2 -H3 2 -H4 2 used for octamer prediction). PELP1 GAR is predicted to be helical when bound to histones and is depicted as electrostatic surface representation. Also see fig. S5. ( I ) AlphaFold3 structural prediction of the human histone octamer bound to two copies of the PELP1 GAR, 2:1 stoichiometry. Exact protein residues used for the prediction are noted in the panel and same as (H). Also see fig. S6.

Techniques: In Vitro, Sequencing, Negative Control, Variant Assay, Filtration, Chromatography, Binding Assay, Migration, SDS Page, Control, Structural Proteomics

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Cartoon of the human rixosome highlighting the SENP3-interacting region. ( B ) N-terminal FLAG-tagged SENP3 truncations (1 to 3) used in the co-IP experiment displayed in (C). Checkmarks and X marks denote qualitative binding results for specific rixosome components in (C) to the SENP3 truncation used for co-IP. ( C ) SDS-PAGE and Western blot using antibodies for the endogenous rixosome proteins identified the presence or absence of rixosome proteins (endogenous and exogenous) upon co-IP with SENP3 truncations. ( D ) X-ray crystal structure of the SENP3 protease domain (311 to 574aa, dark blue) C532S mutant bound to the PELP1 SLiM peptide (764 to 781aa, teal) at 2.93 Å resolution. SENP3 active site with catalytic triad residues is circled in red. ( E ) PELP1 SLiM residues 765 to 767 (FVH motif) and 776 to 778 (VEI motif) form antiparallel β-strand interactions with SENP3 at β6* and β7, respectively. ( F ) The PELP1 SLiM binding interface with the SENP3 protease domain surface. ( G ) The PELP1 FVH motif secondary structure backbone interactions with SENP3 β6*. Putative backbone hydrogen bonding interactions are illustrated by dashed lines. Experimental electron density is displayed as a composite omit map contoured at σ = 0.152 e/Å 3 . ( H ) The PELP1 VEI motif secondary structure backbone interactions with SENP3 β7. Putative backbone hydrogen bonding interactions are illustrated by dashed lines with the experimental density shown as in (G). ( I ) Multiple sequence alignment of PELP1 SLiM and flanking regions. Species abbreviations are the same as . ( J ) Schematic of C-terminal FLAG-tagged PELP1 mutant constructs (1 to 5) used in the co-IP experiment displayed in (K). Checkmarks and X marks denote qualitative binding results for SENP3 (K) to the PELP1 mutant. ( K ) SDS-PAGE and Western blot identified the presence or absence of SENP3 and other rixosome proteins upon co-IP from human cells of PELP1 mutant constructs in (J).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Mutagenesis, Sequencing, Construct

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Size exclusion chromatography (SEC) curves exhibiting the formation of a complex between SENP3 protease domain 302 to 574aa and PELP1 SLiM -containing peptide 761 to 796aa. SDS-PAGE and total protein staining of SEC fractions are displayed below the x axis. ( B ) AlphaFold3 structural models of proSUMO substrates used in endopeptidase cleavage assays. Sequences of the proSUMO C-terminal tails (cleavage sites labeled with arrows) are shown below the structural models. ( C ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM endopeptidase activity assays against proSUMO1, −2, and −3 substrates. Decreasing concentrations (2500 to 0.12 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 5 μM proSUMO substrate. Cleaved proSUMO product is labeled with asterisks. Quantification curves representing percent (%) proSUMO endopeptidase cleavage correspond to the gel images and the enzyme concentration range was 1000 to 15.63 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and total protein stain of in vitro SENP3 (C532A) ± MBP-PELP1 SLiM endopeptidase activity assay, illustrating no activity. ( E ) Quantification curves representing percent (%) proSUMO2 endopeptidase cleavage by SENP protease domain + PELP1 SLiM , SENP3 protease domain alone, or SENP5 protease domain alone during a time course (0 to 450 s). Enzyme concentration was kept constant at 1000 nM along with substrate concentration at 5 μM. Percent cleavage was calculated as in (C). SD was calculated from three independent assay samples ( n = 3). Raw gel images are displayed in fig. S13A. ( F ) Differential scanning fluorimetry curves exhibiting the thermal stabilization of the SENP3 protease domain upon addition of short (“s,” amino acids 764 to 781) and long (“l,” amino acids 764 to 792) PELP1 SLIM peptides. Boltzmann and first derivative curves are shown on the top and bottom of the panel, respectively. Values for change in T m is only shown for the first derivative.

Techniques: Size-exclusion Chromatography, SDS Page, Staining, Labeling, In Vitro, Activity Assay, Incubation, Concentration Assay

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) AlphaFold3 structural model of MBP-Flag-NPM1 (240 to 294aa) SUMO2 conjugate, with the cleavage site indicated with a red arrow. NPM1 SUMOylation site K263 depicted. ( B ) SDS-PAGE and total protein stain of in vitro SENP3 ± MBP-PELP1 SLiM isopeptidase activity assays against MBP-Flag-NPM1-SUMO2 conjugates. Decreasing concentrations (1000 to 3.9 nM) of SENP3 enzyme ± MBP-PELP1 761 to 796aa was incubated for 1 hour at 37°C with 15 μl of on-resin NPM1-SUMO2 conjugates. Cleaved free SUMO product is labeled. Red * on bands in control lanes denote MBP-Flag-NPM1 that copurifies without SUMO2 conjugation. ( C ) Quantification curves representing percent (%) cleavage of substrate over time correspond to the gel images in fig. S13B and the enzyme concentration used was 500 nM. SD was calculated from three independent experiments ( n = 3). ( D ) SDS-PAGE and Western blot of HA-tagged SUMO2 conjugated proteins detected in HEK293FT cell lysate. Changes in amount of HA-SUMO2 conjugated proteins in vivo was assayed after transfecting cells with wild-type or C532A catalytic mutant SENP3 in combination with titrated transfection of wild-type PELP1. Decreasing amounts of transfected DNA expressing exogenous PELP1 with wild-type SENP3 resulted in an overall dose-dependent increase of HA-SUMO2–conjugated proteins similar to that observed with SENP3 C532A (catalytic-dead mutant). ( E ) Same as (D) except HA-tagged SUMO1 was used.

Techniques: SDS Page, Staining, In Vitro, Activity Assay, Incubation, Labeling, Control, Conjugation Assay, Concentration Assay, Western Blot, In Vivo, Mutagenesis, Transfection, Expressing

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Figure Lengend Snippet: ( A ) Swiss army knife model of the rixosome. The PELP1-WDR18-TEX10 scaffolding core is analogous to the housing unit of the knife from which the different diverse tools extend outward, including scissors (RNase PNK RNA cutting), saw blade (SENP3 protease), and corkscrew (MDN1 ATPase motor remodeling). ( B ) Cartoon structural model of the full human rixosome. The scaffolding core (PELP1-WDR18-TEX10) represents the stable core of the rixosome complex from which enzymatic components are connected. The RNA processing complex RNase PNK (LAS1L-NOL9) is physically and flexibly tethered to the stable scaffolding core. The deSUMOylation enzyme SENP3 and AAA-ATPase MDN1 are distinctly integrated with the rixosome through PELP1’s C-terminal IDRs. ( C ) Proposed architectural model of the human rixosome bound to the pre-60S subunit [PDB ID: 8FL2 ]. PELP1’s IDRs organize the enzymatic functions of SENP3 and MDN1. ( D ) Proposed architectural model of the human rixosome bound to polycomb and functioning in gene silencing at facultative heterochromatin. PELP1’s IDRs likely organize multiple enzymatic and nonenzymatic functions at heterochromatin, including SENP3 and MDN1 activity, and putative histone chaperoning by the GAR.

Techniques: Scaffolding, Activity Assay

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Article Snippet: Two microliters of clarified lysate was incubated for 1 hour at 4°C on a nutator with 1 μg of anti-PELP1 antibody (Bethyl labs) or 1 μg of normal rabbit immunoglobulin G (Sigma-Aldrich) for negative control.

Techniques: Cryo-EM Sample Prep, Scaffolding, Variant Assay, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Plasmid Preparation, Control

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: Labeling, Sequencing, Residue, Variant Assay, Construct, Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Lysis, Isolation, Staining, Copurification

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: Structural Proteomics, Binding Assay, Sequencing, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Staining

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: In Vitro, Sequencing, Negative Control, Variant Assay, Filtration, Chromatography, Binding Assay, Migration, SDS Page, Control, Structural Proteomics

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: Co-Immunoprecipitation Assay, Binding Assay, SDS Page, Western Blot, Mutagenesis, Sequencing, Construct

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: Size-exclusion Chromatography, SDS Page, Staining, Labeling, In Vitro, Activity Assay, Incubation, Concentration Assay

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: SDS Page, Staining, In Vitro, Activity Assay, Incubation, Labeling, Control, Conjugation Assay, Concentration Assay, Western Blot, In Vivo, Mutagenesis, Transfection, Expressing

Journal: Science Advances

Article Title: PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex

doi: 10.1126/sciadv.adw4603

Techniques: Scaffolding, Activity Assay

Journal: Cell reports

Article Title: TRMT1L-catalyzed m 2 2 G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress

doi: 10.1016/j.celrep.2024.115167

Figure Lengend Snippet:

Article Snippet: Rabbit anti-PELPI Antibody , Bethyl , Cat#A300-180A; RRID: AB_242526.

Techniques: Recombinant, Membrane, Reverse Transcription, Protease Inhibitor, Magnetic Beads, Staining, Mutagenesis, Silver Staining, BIA-KA, RNA Sequencing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: TRMT1L-catalyzed m 2 2 G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress

doi: 10.1016/j.celrep.2024.115167

Figure Lengend Snippet:

Article Snippet: Rabbit anti-PELPI Antibody , Bethyl , CatA300-180A; RRID: AB_242526.

Techniques: Recombinant, Membrane, Reverse Transcription, Protease Inhibitor, Magnetic Beads, Staining, Mutagenesis, Silver Staining, BIA-KA, RNA Sequencing, Plasmid Preparation, Software

Figure 1. TRMT1L interacts with components of the Rix1 60S biogenesis complex in the nucleolus (A) Mass spectrometry analysis of PELP1-associated complexes. (B and C) (B) Western blot analysis of endogenous PELP1 or (C) TRMT1L immunoprecipitated. Asterisk indicates the presence of the IgG cross-reactivity band. (D) Schematic representation of TRMT1L and TRMT1 proteins. (E) Immunoﬂuorescence detection of endogenous TRMT1L. Scale bar, 20 mm (F) Inhibition of rRNA transcription by CX-5461 impairs TRMT1L nucleolar localization in U2OS cells. Scale bar, 20 mm. See also Figure S1.

Journal: Cell reports

Article Title: TRMT1L-catalyzed m 2 2 G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.

doi: 10.1016/j.celrep.2024.115167

Figure Lengend Snippet: Figure 1. TRMT1L interacts with components of the Rix1 60S biogenesis complex in the nucleolus (A) Mass spectrometry analysis of PELP1-associated complexes. (B and C) (B) Western blot analysis of endogenous PELP1 or (C) TRMT1L immunoprecipitated. Asterisk indicates the presence of the IgG cross-reactivity band. (D) Schematic representation of TRMT1L and TRMT1 proteins. (E) Immunoﬂuorescence detection of endogenous TRMT1L. Scale bar, 20 mm (F) Inhibition of rRNA transcription by CX-5461 impairs TRMT1L nucleolar localization in U2OS cells. Scale bar, 20 mm. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-PELP1 Antibody Bethyl Cat#A300-180A; RRID: AB_242526 Rabbit anti-TRMT1L Antibody Bethyl Cat#A305-120A; RRID: AB_2631515 Mouse anti-TRMT1L Antibody Sigma-Aldrich Cat#SAB1408048; RRID: AB_10741695 Rabbit anti-SENP3 Antibody Cell Signaling Technology Cat#5591; RRID: AB_10694546 Rabbit anti-WDR18 Antibody Castle et al.25 N/A Mouse anti-NPM1 Antibody Santa Cruz Biotechnology Cat#sc-56622; RRID: AB_784888 Mouse anti-uL5 (RPL11) Antibody Sigma-Aldrich Cat#SAB1402896; RRID: AB_10738164 Mouse c-Myc Antibody (9E10) Santa Cruz Biotechnology Cat#sc-40; RRID: AB_627268 Rabbit anti-TRMT1 Antibody Bethyl Cat#A304-205A; RRID: AB_2620402 Rabbit anti-Fibrillarin Antibody Bethyl Cat#A303-891A; RRID: AB_2620241 Rabbit anti-DDX21 Antibody Bethyl Cat#A300-627A; RRID: AB_513601 Rabbit anti-N2,N2-dimethylguanosine (m2,2G) Antibody Abcam Cat#ab211488; RRID: AB_3532174 Rabbit IgG Normal Millipore Cat#NI01; RRID: AB_490574 Mouse anti-GAPDH Antibody Santa Cruz Biotechnology Cat#sc-47724; RRID: AB_627678 Mouse anti-puromycin antibody DSHB Cat#PMY-2A4; RRID: AB_2619605 Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa FluorTM 488 Thermo Fisher Scientific Cat#A-11001; RRID: AB_2534069 Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa FluorTM 594 Thermo Fisher Scientific Cat#A-11012; RRID: AB_2534079 Chemicals, peptides, and recombinant proteins Amersham Hybond N+ nylon membrane GE Healthcare RPN303B AMPure RNAClean XP beads Beckman Coulter A63987 AMV Reverse Transcriptase Promega M510A AMV Reverse Transcriptase 5X Reaction Buffer Promega M515A Aprotinin Fisher BP250310 AEBSF Fisher BP635-500 CX-5461 Cellagen Technologies C2954-2s DMEM (no glucose) Gibco 11966025 Exonuclease I NEB M0293S FastAP Thermosensitive Alkaline Phosphatase Thermo Scientific EF0651 Fetal bovine serum Hyclone SV30014.03 High glucose DMEM HyClone SH30022.01 Immun-Star AP substrate Bio-rad 1705018 Lipofectamine RNAi Max Invitrogen 13778150 Murine RNase inhibitor NEB M0314 Penicillin-Streptomycin Hyclone SH40003.01 Protease inhibitor cocktail Thermo Scientific 78437 Protinase K Invitrogen 46–7185 Protein G Dynabeads Invitrogen 10003D Puromycin Sigma P8833 pCp-Biotin Jena Bioscience NU-1706-Bio (Continued on next page) 18 Cell Reports 44, 115167, January 28, 2025

Techniques: Mass Spectrometry, Western Blot, Immunoprecipitation, Inhibition

PELP1 expression is upregulated in HCC, and high PELP1 expression is associated with poor survival of patients with HCC. Data obtained from TNMplot shows increased expression of PELP1 in patients with HCC ( A ). The results from TCGA-UALCAN database show that PELP1 expression is increased with HCC progression ( B ). The Clinical Proteomic Tumor Analysis Consortium database shows high PELP1 expression in HCC tumor tissues compared with normal tissues ( C ). Association of protein expression of PELP1 with overall survival of patients with HCC was obtained from The Human Protein Atlas ( D ). TMA was used to investigate the PELP1 expression in 86 HCC and six normal liver specimens by IHC and quantified ( E and F ). Scale bar, 100 μm. P values are calculated using the Student t test. ****, p < 0.0001.

Journal: Cancer Research Communications

Article Title: PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

doi: 10.1158/2767-9764.CRC-24-0173

Figure Lengend Snippet: PELP1 expression is upregulated in HCC, and high PELP1 expression is associated with poor survival of patients with HCC. Data obtained from TNMplot shows increased expression of PELP1 in patients with HCC ( A ). The results from TCGA-UALCAN database show that PELP1 expression is increased with HCC progression ( B ). The Clinical Proteomic Tumor Analysis Consortium database shows high PELP1 expression in HCC tumor tissues compared with normal tissues ( C ). Association of protein expression of PELP1 with overall survival of patients with HCC was obtained from The Human Protein Atlas ( D ). TMA was used to investigate the PELP1 expression in 86 HCC and six normal liver specimens by IHC and quantified ( E and F ). Scale bar, 100 μm. P values are calculated using the Student t test. ****, p < 0.0001.

Article Snippet: Two PELP1 antibodies (A300-180A and IHC-00013) used in this study were purchased from Bethyl Laboratories, Inc.

Techniques: Expressing

PELP1 -KD/SMIP34 treatment decreased cell viability, clonogenicity, and invasiveness of HCC cells. PELP1-KD in Huh7 and Hep3B cell lines were confirmed by Western blot ( A ). Cell viability and clonogenic assays were performed to assess the impact of PELP1 KD on the growth of Huh7 and Hep3B cells ( B and C ). The effect of PELP1 -KD on the invasion of HCC cells was performed using Matrigel invasion chamber assays ( D ). The effect of SMIP34 on the cell viability of six HCC cells was measured using MTT assay ( E ). Effect of SMIP34 (5 μmol/L) on clonogenic cell survival was examined using colony formation assay ( F ). The effect of SMIP34 (10 μmol/L) treatment on HCC cells invasion was performed using Matrigel invasion chamber assay ( G ). The results show the mean ± SEM, n = 3. P values are calculated using the Student t test and one-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Journal: Cancer Research Communications

Article Title: PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

doi: 10.1158/2767-9764.CRC-24-0173

Figure Lengend Snippet: PELP1 -KD/SMIP34 treatment decreased cell viability, clonogenicity, and invasiveness of HCC cells. PELP1-KD in Huh7 and Hep3B cell lines were confirmed by Western blot ( A ). Cell viability and clonogenic assays were performed to assess the impact of PELP1 KD on the growth of Huh7 and Hep3B cells ( B and C ). The effect of PELP1 -KD on the invasion of HCC cells was performed using Matrigel invasion chamber assays ( D ). The effect of SMIP34 on the cell viability of six HCC cells was measured using MTT assay ( E ). Effect of SMIP34 (5 μmol/L) on clonogenic cell survival was examined using colony formation assay ( F ). The effect of SMIP34 (10 μmol/L) treatment on HCC cells invasion was performed using Matrigel invasion chamber assay ( G ). The results show the mean ± SEM, n = 3. P values are calculated using the Student t test and one-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Two PELP1 antibodies (A300-180A and IHC-00013) used in this study were purchased from Bethyl Laboratories, Inc.

Techniques: Western Blot, MTT Assay, Colony Assay, Invasion Chamber Assay

Analysis of global transcriptional changes in PELP1 -KD HCC cells. Volcano plot of differentially expressed genes with PELP1-KD in Hep3B cells is displayed ( n = 3; A ). PELP1-downregulated pathways were identified using differentially expressed gene signature ( B ). Gene set enrichment analysis plots show negatively enriched pathways by PELP1-KD ( C ). Heatmap images of the specific genes affected by PELP1 -KD ( D ). RT-qPCR was used to validate the specific genes that were differentially regulated by PELP1 -KD in RNA-seq analysis ( E ). Data are represented as the mean ± SEM. P values are calculated using two-way ANOVA, ****, p < 0.0001. ES, enrichment score.

Journal: Cancer Research Communications

Article Title: PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

doi: 10.1158/2767-9764.CRC-24-0173

Figure Lengend Snippet: Analysis of global transcriptional changes in PELP1 -KD HCC cells. Volcano plot of differentially expressed genes with PELP1-KD in Hep3B cells is displayed ( n = 3; A ). PELP1-downregulated pathways were identified using differentially expressed gene signature ( B ). Gene set enrichment analysis plots show negatively enriched pathways by PELP1-KD ( C ). Heatmap images of the specific genes affected by PELP1 -KD ( D ). RT-qPCR was used to validate the specific genes that were differentially regulated by PELP1 -KD in RNA-seq analysis ( E ). Data are represented as the mean ± SEM. P values are calculated using two-way ANOVA, ****, p < 0.0001. ES, enrichment score.

Article Snippet: Two PELP1 antibodies (A300-180A and IHC-00013) used in this study were purchased from Bethyl Laboratories, Inc.

Techniques: Quantitative RT-PCR, RNA Sequencing

SMIP34 treatment blocked PELP1-mediated extranuclear signaling and decreased global protein synthesis. Huh7, SNU398, Hep3B, and SNU423 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L) to examine PELP1 degradation and the stability of the Rix1 complex by Western blotting ( A and C ). Hep3B, Huh7, and SNU449 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of known PELP1 downstream signaling targets were analyzed by Western blotting ( B ). Hep3B and Huh7 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of E2F1 and c-Myc expressions was analyzed by Western blotting ( D ). Hep3B and Huh7 cells treated with SMIP34 (5, 10, and 15 μm) or Hep3B and Huh7 cells with PELP1-KD were subjected to 30 miniutes of incubation with puromycin (1 μmol/L). The effect of this treatment on global protein synthesis was measured by Western blotting using an anti-puromycin antibody ( E and F ).

Journal: Cancer Research Communications

Article Title: PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

doi: 10.1158/2767-9764.CRC-24-0173

Figure Lengend Snippet: SMIP34 treatment blocked PELP1-mediated extranuclear signaling and decreased global protein synthesis. Huh7, SNU398, Hep3B, and SNU423 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L) to examine PELP1 degradation and the stability of the Rix1 complex by Western blotting ( A and C ). Hep3B, Huh7, and SNU449 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of known PELP1 downstream signaling targets were analyzed by Western blotting ( B ). Hep3B and Huh7 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of E2F1 and c-Myc expressions was analyzed by Western blotting ( D ). Hep3B and Huh7 cells treated with SMIP34 (5, 10, and 15 μm) or Hep3B and Huh7 cells with PELP1-KD were subjected to 30 miniutes of incubation with puromycin (1 μmol/L). The effect of this treatment on global protein synthesis was measured by Western blotting using an anti-puromycin antibody ( E and F ).

Article Snippet: Two PELP1 antibodies (A300-180A and IHC-00013) used in this study were purchased from Bethyl Laboratories, Inc.

Techniques: Western Blot, Incubation

PELP1 -KD or SMIP34 treatment suppresses HCC xenograft tumor growth in vivo . Hep3B–control and Hep3B– PELP1 -KD model cells were injected subcutaneously into female ( A , D , and G ) or male SCID mice ( B , E , and H ). Tumor volumes were assessed at 3 to 5 days of intervals. Tumor volume ( A and B ), tumor weights ( D and E ), and IHC analyses of Ki67 and PELP1 ( G and H ) for tumors are shown. Hep3B xenograft tumor fragments measuring 2 to 3 mm 3 were implanted subcutaneously , and following tumor establishment (∼200 mm 3 ), the mice were randomly assigned to receive either vehicle (control) or SMIP34 (20 mg/kg body weight) 5 days a week through i.p. injection ( n = 5). Tumor volume ( C ), tumor weights ( F ) and IHC analyses of Ki67 and PELP1 ( I ) for tumors are shown. Scale bar, 100 μm. Data are represented as the mean ± SEM. P values are calculated using Student t test and two-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Cont sh (Control short hairpin).

Journal: Cancer Research Communications

Article Title: PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

doi: 10.1158/2767-9764.CRC-24-0173

Figure Lengend Snippet: PELP1 -KD or SMIP34 treatment suppresses HCC xenograft tumor growth in vivo . Hep3B–control and Hep3B– PELP1 -KD model cells were injected subcutaneously into female ( A , D , and G ) or male SCID mice ( B , E , and H ). Tumor volumes were assessed at 3 to 5 days of intervals. Tumor volume ( A and B ), tumor weights ( D and E ), and IHC analyses of Ki67 and PELP1 ( G and H ) for tumors are shown. Hep3B xenograft tumor fragments measuring 2 to 3 mm 3 were implanted subcutaneously , and following tumor establishment (∼200 mm 3 ), the mice were randomly assigned to receive either vehicle (control) or SMIP34 (20 mg/kg body weight) 5 days a week through i.p. injection ( n = 5). Tumor volume ( C ), tumor weights ( F ) and IHC analyses of Ki67 and PELP1 ( I ) for tumors are shown. Scale bar, 100 μm. Data are represented as the mean ± SEM. P values are calculated using Student t test and two-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Cont sh (Control short hairpin).

Article Snippet: Two PELP1 antibodies (A300-180A and IHC-00013) used in this study were purchased from Bethyl Laboratories, Inc.

Techniques: In Vivo, Control, Injection

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