sumo 1 Search Results


sumo1  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc sumo1
Fig. 1 deSUMO2/3-ylation induces mitophagy and promotes HIM. A Hypoxia induces mitophagy. HeLa cells were transfected with Mito- pHfluorin per 35mm-dish and exposed to normoxia (N) or hypoxia (H;1% O2 for 4, 8, 16, or 24 h) (Scale bar, 10 μm). Upper panel shows that hypoxia-induced mitophagy is detectable as early as 4 h after cells exposed to 1% O2. Histogram in the right panel shows the average number of puncta per cell under indicated time points (n = 19 ~ 51 cells; *p < 0.05; **p < 0.01; ***p < 0.001; ****; unpaired t-test). B, C Hypoxia causes decreased SUMO2/3-ylation (B, n = 5, biological replicates; *p < 0.05; paired t-test) but not <t>SUMO1-ylation</t> (C, n = 6, biological replicates; N.S., non-significant; paired t-test) in HeLa cells. HeLa cells were exposed to 1% O2 for 24 h. Whole cell lysate samples were prepared and blotted as indicated.
Sumo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SUMO-1+Antibody/pm39638786-241-40-44
Average 96 stars, based on 1 article reviews
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pcdna3 ha sumo1  (Addgene inc)
92
Addgene inc pcdna3 ha sumo1

Pcdna3 Ha Sumo1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/pcDNA3+HA-Sumo1+WT+(Plasmid+%2348966)/pmc08710555-71-0-4
Average 92 stars, based on 1 article reviews
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pcdna3 1 ha sumo 1  (Addgene inc)
92
Addgene inc pcdna3 1 ha sumo 1

Pcdna3 1 Ha Sumo 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/pcDNA3-HA-Sumo1+(Plasmid+%2321154)/pmc05728694-446-38-47
Average 92 stars, based on 1 article reviews
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anti sumo1 antibody  (Proteintech)
95
Proteintech anti sumo1 antibody
Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of <t>SUMO1/2/3</t> protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).
Anti Sumo1 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SUMO1+Antibody/pmc11226984-15-0-3
Average 95 stars, based on 1 article reviews
anti sumo1 antibody - by Bioz Stars, 2026-10
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anti sumo 1  (Santa Cruz Biotechnology)
94
Santa Cruz Biotechnology anti sumo 1
Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of <t>SUMO1/2/3</t> protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).
Anti Sumo 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SUMO-1+Antibody/10__1074_slash_jbc__m603772200-55-17-21
Average 94 stars, based on 1 article reviews
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pmrfp expression vector  (Addgene inc)
85
Addgene inc pmrfp expression vector
Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of <t>SUMO1/2/3</t> protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).
Pmrfp Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/pmRFP-SUMO1+GG+(Plasmid+%2313990)/pmc04743023-309-21-24
Average 85 stars, based on 1 article reviews
pmrfp expression vector - by Bioz Stars, 2026-10
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fauke mechior  (Addgene inc)
93
Addgene inc fauke mechior
Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of <t>SUMO1/2/3</t> protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).
Fauke Mechior, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/pET11a-SUMO1+(Plasmid+%2353138)/bio_rxiv__2022__10__12__511930-170-6-8
Average 93 stars, based on 1 article reviews
fauke mechior - by Bioz Stars, 2026-10
93/100 stars
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anti senp3 polyclonal antibody  (Proteintech)
94
Proteintech anti senp3 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 (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 <t>SENP3</t> (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 Senp3 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SENP3+Antibody/pmc12292926-347-95-111
Average 94 stars, based on 1 article reviews
anti senp3 polyclonal antibody - by Bioz Stars, 2026-10
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ripa buffer  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology ripa buffer
( 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 <t>SENP3</t> (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.
Ripa Buffer, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SUMO-1+siRNA/pm38587211-66-7-9
Average 93 stars, based on 1 article reviews
ripa buffer - by Bioz Stars, 2026-10
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peyfp sumo1  (Addgene inc)
91
Addgene inc peyfp sumo1
( 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 <t>SENP3</t> (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.
Peyfp Sumo1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/pEYFP+SUMO-1+(Plasmid+%2313380)/pm30510112-320-16-17
Average 91 stars, based on 1 article reviews
peyfp sumo1 - by Bioz Stars, 2026-10
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ha sumo 1  (Addgene inc)
91
Addgene inc ha sumo 1
( 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 <t>SENP3</t> (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.
Ha Sumo 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sumo+1/SRa-HA-SUMO1+(Sentrin+1)+(Plasmid+%2317359)/pmc03346107-231-6-5
Average 91 stars, based on 1 article reviews
ha sumo 1 - by Bioz Stars, 2026-10
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Image Search Results


Fig. 1 deSUMO2/3-ylation induces mitophagy and promotes HIM. A Hypoxia induces mitophagy. HeLa cells were transfected with Mito- pHfluorin per 35mm-dish and exposed to normoxia (N) or hypoxia (H;1% O2 for 4, 8, 16, or 24 h) (Scale bar, 10 μm). Upper panel shows that hypoxia-induced mitophagy is detectable as early as 4 h after cells exposed to 1% O2. Histogram in the right panel shows the average number of puncta per cell under indicated time points (n = 19 ~ 51 cells; *p < 0.05; **p < 0.01; ***p < 0.001; ****; unpaired t-test). B, C Hypoxia causes decreased SUMO2/3-ylation (B, n = 5, biological replicates; *p < 0.05; paired t-test) but not SUMO1-ylation (C, n = 6, biological replicates; N.S., non-significant; paired t-test) in HeLa cells. HeLa cells were exposed to 1% O2 for 24 h. Whole cell lysate samples were prepared and blotted as indicated.

Journal: Cell death & disease

Article Title: SENP3-FIS1 axis promotes mitophagy and cell survival under hypoxia.

doi: 10.1038/s41419-024-07271-8

Figure Lengend Snippet: Fig. 1 deSUMO2/3-ylation induces mitophagy and promotes HIM. A Hypoxia induces mitophagy. HeLa cells were transfected with Mito- pHfluorin per 35mm-dish and exposed to normoxia (N) or hypoxia (H;1% O2 for 4, 8, 16, or 24 h) (Scale bar, 10 μm). Upper panel shows that hypoxia-induced mitophagy is detectable as early as 4 h after cells exposed to 1% O2. Histogram in the right panel shows the average number of puncta per cell under indicated time points (n = 19 ~ 51 cells; *p < 0.05; **p < 0.01; ***p < 0.001; ****; unpaired t-test). B, C Hypoxia causes decreased SUMO2/3-ylation (B, n = 5, biological replicates; *p < 0.05; paired t-test) but not SUMO1-ylation (C, n = 6, biological replicates; N.S., non-significant; paired t-test) in HeLa cells. HeLa cells were exposed to 1% O2 for 24 h. Whole cell lysate samples were prepared and blotted as indicated.

Article Snippet: The primary antibodies used were: FIS1 (rabbit polyclonal Ab; Proteintech 10956-1-AP; 1:100 dilution), FKBP8 (mouse monoclonal Ab; Santa Cruz Biotechnology sc-166607, 1:200 dilution), SENP1 (recombinant monoclonal Ab; Abcam EPR3844, 1:200 dilution), SENP3 (rabbit monoclonal Ab; Cell Signaling #5591, 1:500 dilution), SUMO1 (rabbit polyclonal Ab; Cell Signaling #4930, 1:250 dilution), SUMO2/3 (rabbit monoclonal Ab; Cell Signaling #4971, 1:100 dilution), TBC1D15 (rabbit polyclonal Ab; ProteinTech, 1:100 dilution), and TBC1D17 (rabbit polyclonal Ab; ProteinTech, 1:100 dilution).

Techniques: Transfection

Journal: iScience

Article Title: Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

doi: 10.1016/j.isci.2021.103484

Figure Lengend Snippet:

Article Snippet: pcDNA3 HA-SUMO1 , , Addgene plasmid # 48966.

Techniques: Recombinant, Blocking Assay, cDNA Synthesis, Plasmid Preparation, Western Blot, Cell Culture, Mutagenesis, Software

Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of SUMO1/2/3 protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet: Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of SUMO1/2/3 protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Inhibition, Control, Western Blot, Incubation

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet:

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Plasmid Preparation, Extraction, Microarray, Software

( 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.

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

( 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.

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.

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: 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 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).

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).

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: 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.

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.

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: 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.

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.

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: 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.

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.

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: Scaffolding, Activity Assay