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    Structured Review

    Bethyl bms1
    Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or <t>BMS1</t> (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
    Bms1, supplied by Bethyl, 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/a305+109a+t/BMS1%2FBMS1L+Antibody/pm38427561-196-32-33
    Average 92 stars, based on 1 article reviews
    bms1 - by Bioz Stars, 2026-10
    92/100 stars

    Images

    1) Product Images from "SARS-CoV-2 targets ribosomal RNA biogenesis."

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.113891

    Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
    Figure Legend Snippet: Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

    Techniques Used: Staining, RNA Immunoprecipitation, Binding Assay, Two Tailed Test

    Related Articles

    Recombinant:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Western Blot:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Fluorescence:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Transfection:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Reverse Transcription:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Membrane:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A

    Protease Inhibitor:

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-mouse IgG HRP linked secondary Cytiva Cat# NA931; RRID: AB_772210 Anti-rabbit IgG HRP linked secondary Cytiva Cat# NA934; RRID: AB_2722659 BMS1 Bethyl Cat# A305-109A-T; RRID: AB_2631504 BMS1 Santa Cruz Cat# sc-271040; RRID: AB_10611468 FBL Abcam Cat# Ab4566; RRID: AB_304523 FBL Abcam Cat# Ab226178 FCF1 Invitrogen Cat# PA5-95887; RRID: AB_2807689 FLAG Abcam Cat# Ab205606; RRID: AB_2916341 FLAG clone M2 Sigma Cat# F1804; RRID: AB_262044 Goat anti-mouse or anti-rabbit 488 or 568 secondary antibodies Invitrogen Cat# A-10680, RRID: AB_2534062; Cat# A-11031, RRID: AB_144696; Cat# A-11008, RRID: AB_143165; Cat# A-11011, RRID: AB_143157 LDHA Millipore Cat# MABC150 NOP56 Bethyl Cat# A302-721A-M; RRID: AB_10755122 NPM1 Sigma Cat# B0556; RRID: AB_2154872 RNA Pol I Invitrogen Cat# PA5-65577; RRID: AB_2662301 RNA Pol I Cell Signaling Cat# 24799S; RRID: AB_2798884 RPS6 (5G10) Cell Signaling Cat# 2217; RRID: AB_331355 UBF Santa Cruz Cat# sc-13125; RRID: AB_671403 V5 (E10/V4RR) Invitrogen Cat# MA515253; RRID: AB_10977225 VCL Sigma Cat# V9131; RRID: AB_477629 Chemicals, peptides, and recombinant proteins Acrylamide/bis-acrylamide solution 37.5:1 Biorad 1610148 Clarity Western ECL substrate Biorad 1705060 Dako fluorescence mounting medium Agilent S302380-2 Denhardt’s Solution Invitrogen 750018 DIRECT-ZOL RNA MICROPREP COLUMNS Zymo Research R2061 Lipofectamine 3000 transfection Reagent Invitrogen L3000015 PageRuler/PageRuler plus prestained ladders Thermo Scientific PI26616, PI26619 Protein A Dynabeads Invitrogen 10006D Quick Start Bradford 1X Dye Reagent BioRad 5000205 RNeasy Mini Kit Qiagen 74106 Sodium hypochlorite Bioshop SYP001 SUPERaseIn RNase Inhibitor Thermo Fisher AM2694 TRIzol Reagent Invitrogen 15596018 M-MLV reverse transcriptase Thermo Scientific 28025013 SensiFAST SYBR No-ROX Bioline BIO-98050 T4 polynucleotide kinase NEB M0201S Turbo DNase Invitrogen AM2238 Microspin G-25 columns Cytiva 27532501 Hybond N+ membrane Cytiva RPN203B Salmon sperm DNA Thermo Fisher 15632011 cOmplete EDTA-free protease inhibitor Thermo Fisher 11873580001 (Continued on next page) Cell Reports 43, 113891, March 26, 2024 11 .. REAGENT or RESOURCE SOURCE IDENTIFIER Critical commercial assays Invitrogen Click-iT Plus OPP Alexa Fluor 488 protein synthesis assay kit Thermo Fisher C10456 Deposited data RNA-seq data from SARS-CoV-2-infected cells (Finkel et al.)4 GEO: GSE162323 RNA-seq data from MERS-CoV-infected cells (Zhang et al.)14 GEO: GSE139516 Experimental models: Cell lines Calu-3 Raught Lab N/A DH5a Mekhail Lab N/A HEK293T Palazzo Lab N/A Oligonucleotides See Table S1 for primers used for PCR Thermo Fisher N/A See Table S1 for the Northern probes used Thermo Fisher N/A Random hexamer primers Thermo Scientific S0142 Recombinant DNA N protein pGBW-m4134545 Benjie Chen, Ginkgo Bioworks Addgene 152594 pCG N protein SARS-CoV2 C-V5-tag (Hayn et al.)58 Addgene 179979 Nsp8 pGBW-m4134010 Benjie Chen, Ginkgo Bioworks Addgene 152437 WT Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175412 K164A/H165A Nsp1 pCDNA4TO-3XFLAG Halo (Kamitani et al.)20 Addgene 175421 Empty pCG (Tanaka et al.)59 Addgene 51476 Software and algorithms BEDTools version 2.26.0 (Quinlan et al.)60 N/A Bowtie2 (Langmead et al.)61 N/A ggplot2 (Wickham et al.)62 N/A SAMTools version 1.7 (Li et al.)63 N/A



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    Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

    Journal: Cell reports

    Article Title: SARS-CoV-2 targets ribosomal RNA biogenesis.

    doi: 10.1016/j.celrep.2024.113891

    Figure Lengend Snippet: Figure 3. Nsp1 localizes to the nucleolus and binds pre-rRNA (A) Localization of Nsp1 and nucleolar UBF relative to DAPI-depleted nucleoli in HEK293T cells. Note the nucleolar localization of Nsp1 visually (images) and in quantifications of signals along dashed line traces (graph). Scale bars, 5 mm (white) and 1 mm (magenta). (B)Immunoblottingofwhole-cell(WC),nuclear/cytoplasmic(Nuc./Cyto.),ornucleolarfractionsfromHEK293Tcellstransfectedwithemptyvector(EV)orFLAG-tagged Nsp1 or Nsp1-KH. Nucleolar protein-56 (NOP56) and FBL mark nucleoli, lactate dehydrogenase A (LDHA) is cytoplasmic, and Ponceau-stained proteins are controls. (C–E) RNA immunoprecipitation pulling down FLAG-tagged Nsp1 (C) or endogenous FBL (D) or BMS1 (E). (F) RNA immunoprecipitation measuring endogenous FBL or BMS1 binding to U3 snoRNA. (C–F) Data are mean +SD from n = 4 (C and D) or n = 3 (E and F) biological replicates, except n = 2 for BMS1 in (F), and were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (C) or two-tailed unpaired t tests (D–F). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

    Article Snippet: Samples were separated using 10% acrylamide/bis solution 37.5:1 (Biorad) gels and analyzed by immunoblotting using antibodies against FLAG (Sigma), RNA Pol I (Cell Signaling or Invitrogen), NPM1 (Sigma), FCF1 (Invitrogen), VCL (Sigma), BMS1 (Bethyl), FBL (Abcam), NOP56 (Bethyl), V5 (Invitrogen), RPS6 (Cell Signaling).

    Techniques: Staining, RNA Immunoprecipitation, Binding Assay, Two Tailed Test