Recombinant:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
Produced:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
Protease Inhibitor:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
Saline:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
Luciferase:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
Mutagenesis:Article Title: G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Polyclonal rabbit anti-DDX3X Abcam Cat# ab235940; RRID:AB_2910140 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21206; RRID:AB_2535792 Chemicals, peptides, and recombinant proteins DDX3X(WT)-GFP This paper N/A DDX3X(WT) This paper N/A DDX3X(G325E)-GFP This paper N/A DDX3X(D354V)-GFP This paper N/A G3BP1 This Paper N/A GFP-G3BP1 Guillén-Boixet et al.14 N/A GFP-G3BP1-DNTF Guillén-Boixet et al.14 N/A Aminoallyl-UTP-Cy5 Jena Bioscience Cat# NU-821-CY5 Benzonase Produced in-house N/A cOmplete, EDTA-free Protease Inhibitor Cocktail Roche Cat# 05056489001 RNase A Roth Cat# 7156.1 RNase If NEB Cat# M0243S His-3C protease Produced in-house N/A RNaseOUT Invitrogen Cat# 10777019 Pyruvate Kinase/Lactic Dehydrogenase (PK/LDH) Sigma-Aldrich Cat# P0294 Sodium hydrogen arsenate heptahydrate, ASC, 98.0-102.0% Alfa Aesar Cat# 33373 ESF 921 Expression systems Cat# 96-001-01 Amylose Resin NEB Cat# E8021L Amylose Resin High Flow NEB Cat# E8022L lithium chloride Thermo Fischer Scientific Cat# AM1344 2 x RNA loading dye Thermo Fischer Scientific Cat# R0641 DMEM (1x) + high Glucose and no phenolred Gibco Cat# 31053028 DMEM (1x) + GlutaMAX Gibco Cat# 10566016 RPMI 1640 Gibco Cat#11875093 RPMI 1640 (-Methionine) Gibco Cat# A1451701 20X saline-sodium citrate Invitrogen Cat#15557036 dextran sulfate (MW > 500,000) Merck Cat#S4030 formamide Merck Cat#47671 RK-33 Selleck Chemicals Cat# S8246 DAPI Fluoromount-G SouthernBiotech Cat# 0100-20 Critical commercial assays mMESSAGE mMACHINE T7 Transcription Kit Thermo Fisher Scientific Cat# AM1344 Nano-Glo Luciferase Assay Promega Cat# N1110 Q5 Site-Directed Mutagenesis Kit NEB Cat# E0554S RNeasy Mini kit Quiagen Cat# 74104 Rabbit Reticulocyte Lysate System, Nuclease treated Promega Cat# L4960 Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10428 (Continued on next page) e1 Molecular Cell 85, 1–17.e1–e11, February 6, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Lipofectamine 3000 Invitrogen Cat#2757099 Escort IV transfection reagent Sigma-Aldrich Cat’ L-3287 Experimental models: Organisms/strains Hela Kyoto Hyman lab N/A U2OS G3BP1/2 knock out expressing G3BP1-mCherry Hyman lab N/A U2OS G3BP1/2 knock out Kedersha et al.15; Laboratory of Paul Anderson N/A SF9 ESF Expression systems Cat# 94-001F Oligonucleotides 35-nt RNAATTO590 (AUCUAGUACAUGUCUAGUCAGUAUCUAGU GAUUA) IDT N/A NRAS-NLuc- mRNA GGGGCCGGAAGUGCCGCUCCUUGGUGG GGGCUGUUCAUGGCGGUUCCGGGGUCUCCAACAUUUUUCC CGGCUGUGGUCCUAAAUCUGUCCAAAGCAGAGGCAGUGGAG CUUGAGGUUCUUGCUGGUGUGAACUGCAGGCCGCCACCAUG GUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAAC UGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCU AUUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAU UGAUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUG AUCAAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUC CUGUGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGU ACUUUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUA UUUUGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGU AAAAAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAA GAUUAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAU UAUUUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUA UGUGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGC UCGAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat#219990 (Adgene) Beta-globin-NLuc- mRNA ACAUUUGCUUCUGACACAACUGUGU UCACUAGCAACCUCAAACAGACACCCUGCAGGCCGCCACCAU GGUUUUUACUUUAGAAGAUUUUGUUGGUGAUUGGCGUCAAA CUGCUGGUUAUAAUUUAGAUCAAGUUUUAGAACAAGGUGGUG UUUCUUCUUUGUUUCAAAACUUAGGUGUUUCUGUUACUCCUA UUCAACGUAUUGUUUUAUCUGGUGAAAAUGGUUUGAAGAUUG AUAUUCAUGUUAUUAUUCCAUAUGAAGGUUUGUCUGGUGAUC AAAUGGGUCAAAUUGAAAAGAUUUUCAAAGUUGUUUAUCCUG UGGACGAUCAUCAUUUCAAAGUUAUUUUGCAUUAUGGUACU UUAGUUAUUGAUGGUGUUACUCCUAAUAUGAUUGAUUAUUU UGGUCGUCCUUAUGAAGGUAUUGCUGUUUUCGAUGGUAAA AAGAUCACUGUUACUGGUACUUUAUGGAAUGGUAACAAGAU UAUUGAUGAACGUUUGAUUAAUCCUGAUGGUUCUUUAUUAU UUCGUGUUACUAUUAAUGGUGUUACUGGUUGGCGUUUAUG UGAACGUAUUUUAGCUUAAGCGGCCGCACCGGUGCUAGCUC GAGACUAGUCGACAAAAAAAAAAAAAAAAAAAAAAA This paper Cat# 219989 (Adgene) hp-RNA35: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU GAA UAG UGA UUC AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A G13A-C24U mutant hp-RNA35-mut: 5’ - ATTO 647N - C6 - UGU UGA ACU UUU AAA UAG UGA UUU AGG AGG UUA AU - C6 - ATTO 532 - 3’ IBA N/A hp-RNA60: 5’ - TYE665 - AAA UAC AUG UCU AGU CAG UAU CUA GUG AUU UAG UAC AUG UCU AGU CAG UAU CUA GUG CCC - Alexa Fluor 532 - 3 IDT N/A polyA Roche Cat# 10108626001 (Continued on next page) Molecular Cell 85, 1–17.e1–e11, February 6, 2025 e2
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