Recombinant:
Article Title: Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins MG132 Peptide Institute Cat # 3175-v Puromycin FUJIFILM Wako Cat # 160-23151 Cycloheximide FUJIFILM Wako Cat # 037-20991 Nocodazole FUJIFILM Wako Cat # 140-08531 Doxycycline Sigma-Aldrich Cat #D9891 poly-L-lysine solution Sigma-Aldrich Cat #P8920 MLN8054 AdooQ Bioscience Cat # A10601 Protease inhibitor cocktail Sigma-Aldrich Cat #P8340 Phosphatase inhibitor Cocktail Nacalai Tesque Cat # 07574-61 Polyethylenimine MAX Polysciences Cat # 24765-1 TransIT-X2 Dynamic Delivery System Mirus Bio Cat # MIR6000 Protein G-Sepharose 4 Fast Flow Cytiva Cat # 17-0618-01 Ab-Capcher Protein A-R28 beads ProteNova Cat # P-002-10 anti-DYKDDDDK tag antibody beads FUJIFILM Wako Cat # 012-22781 Glutathione Sepharose 4B beads Cytiva Cat # 17-0756-01 Ni-NTA-agarose Thermo Fisher Scientific Cat #R90115 HA-Peptide Roche Cat # 11-666-975-001 Ubiquitin Sigma-Aldrich Cat #U6253 Recombinant Human Ubiquitin-Activating Enzyme (UBE1) Bio-Techne Cat # E 305-025 Recombinant Human His-UBE2N/MMS2 complex Bio-Techne Cat #E2-666-100 Ubiquitin Conjugation Reaction buffer Kit Bio-Techne Cat # SK-10 Phos-tag Acrylamide FUJIFILM Wako Cat # 304-93521 Acrylamide/Bisacrylamide (37.5:1) FUJIFILM Wako Cat # 018-25625 Wide range gel preparation buffer Nacalai Tesque Cat # 07831-94 Zinc nitrate hexahydrate Sigma-Aldrich Cat # 10196-18-6 His-Nus This paper N/A His-Nus-Aurora A-WT, F31I, K162M This paper N/A His-Nus-NEK2 This paper N/A GST This paper N/A GST-OLA1-WT, T124A, T124E, T124E + DM This paper N/A GST-Aurora A-N, DN This paper N/A His-OLA1 This paper N/A Critical commercial assays GenomeLab Human STR Primer set Beckman Coulter Cat # A20100 SilencerTM siRNA construction Kit Thermo Fisher Scientific Cat # AM1620 Experimental models: Cell lines HeLa ATCC Cat # CCL-2; RRID: CVCL_0030 HEK-293T ATCC Cat # CRL-11268 U2OS ATCC Cat # HTB-96 RRID: CVCL_0042 MCF7 ATCC Cat # HTB-22 HeLa-tet-shOLA1 This paper N/A Oligonucleotides siRNA targeting sequence Aurora A-1: 50-AUUCUUCCCAGCGCGUUCC-30 Hirota et al.65 N/A (Continued on next page) Cell Reports 42, 112850, August 29, 2023 21 .. REAGENT or RESOURCE SOURCE IDENTIFIER siRNA targeting sequence Aurora A-2: 50-AUGCCCUGUCUUACUGUCA-30 Kufer et al.66 N/A siRNA targeting sequence Aurora A-3: Integrated DNA Technologies Cat # hs.Ri.AURKA.13.1 Recombinant DNA pCY4B-FLAG-Aurora A This paper N/A pCY4B-FLAG-Aurora A-mutant This paper N/A pCY4B-FLAG-1–133 (N) This paper N/A pCY4B-FLAG-105–403 (DN) This paper N/A pCY4B-HA-Aurora A Yoshino et al.25 N/A pET-Aurora A Yoshino et al.25 N/A pET-Aurora A-mutant This paper N/A pEGFP-Aurora A This paper N/A pEGFP-Aurora A-mutant This paper N/A pEGFP-Aurora A-1–133 (N) This paper N/A pEGFP-Aurora A-105–403 (DN) This paper N/A pGEX-Aurora A-1–133 (N) This paper N/A pGEX-Aurora A-105–403 (DN) This paper N/A pCY4B-HA-OLA1 Matsuzawa et al.16 N/A pCY4B-HA-OLA1-Mutant Yoshino et al.21 This paper N/A pCY4B-HA-OLA1-1–171 (N) This paper N/A pCY4B-HA-OLA1-166–396 (C) This paper N/A pCY4B-FLAG-OLA1 Matsuzawa et al.16 N/A pCY4B-FLAG-OLA1-Mutant This paper N/A pGEX-OLA1 Matsuzawa et al.16 N/A pGEX-OLA1-Mutant Yoshino et al.21 This paper N/A pET-OLA1 Matsuzawa et al.16 N/A pLKO-tet-shOLA1-puro This paper N/A pCMV-Myc-ubiquitin Starita et al.67 N/A pCMV-Myc-ubiquitin-mutant This paper N/A pEGFP-centrin Yoshino et al.21 N/A pEGFP-NEK2 This paper N/A pEGFP-NEK2-K37R This paper N/A pET-NEK2 This paper N/A pCY4B-HA-NEK2 This paper N/A pCY4B-HA-NEK2-K37R This paper N/A psPAX2 Addgene Cat # 12260 pMD2.G Addgene Cat # 12259 EZ-Tet-pLKO-Puro Addgene Cat # 85966 pCY4B-FLAG-mCherry-puro This paper N/A Software and algorithms ImageJ National Institute of Health https://ImageJ.nih.gov/ij/ JMP 12 software JMP https://www.jmp.com/ja_jp/software.html Other ImageQuant LAS 4000mini Cytiva N/A BZ-X710 Keyence N/A 22 Cell Reports 42, 112850, August 29, 2023
Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation
Article Snippet: .. Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025). .. Cells were collected, quickly rinsed with 1× PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 1.5 mM MgCl 2 , 2 mM EGTA, 2 mM DTT and 1× cOmplete Protease Inhibitor Cocktail (Roche)) on ice for 30 min and centrifuged at 12,000 rpm, 4 °C for 10 min.
Ubiquitin Proteomics:
Article Title: Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins MG132 Peptide Institute Cat # 3175-v Puromycin FUJIFILM Wako Cat # 160-23151 Cycloheximide FUJIFILM Wako Cat # 037-20991 Nocodazole FUJIFILM Wako Cat # 140-08531 Doxycycline Sigma-Aldrich Cat #D9891 poly-L-lysine solution Sigma-Aldrich Cat #P8920 MLN8054 AdooQ Bioscience Cat # A10601 Protease inhibitor cocktail Sigma-Aldrich Cat #P8340 Phosphatase inhibitor Cocktail Nacalai Tesque Cat # 07574-61 Polyethylenimine MAX Polysciences Cat # 24765-1 TransIT-X2 Dynamic Delivery System Mirus Bio Cat # MIR6000 Protein G-Sepharose 4 Fast Flow Cytiva Cat # 17-0618-01 Ab-Capcher Protein A-R28 beads ProteNova Cat # P-002-10 anti-DYKDDDDK tag antibody beads FUJIFILM Wako Cat # 012-22781 Glutathione Sepharose 4B beads Cytiva Cat # 17-0756-01 Ni-NTA-agarose Thermo Fisher Scientific Cat #R90115 HA-Peptide Roche Cat # 11-666-975-001 Ubiquitin Sigma-Aldrich Cat #U6253 Recombinant Human Ubiquitin-Activating Enzyme (UBE1) Bio-Techne Cat # E 305-025 Recombinant Human His-UBE2N/MMS2 complex Bio-Techne Cat #E2-666-100 Ubiquitin Conjugation Reaction buffer Kit Bio-Techne Cat # SK-10 Phos-tag Acrylamide FUJIFILM Wako Cat # 304-93521 Acrylamide/Bisacrylamide (37.5:1) FUJIFILM Wako Cat # 018-25625 Wide range gel preparation buffer Nacalai Tesque Cat # 07831-94 Zinc nitrate hexahydrate Sigma-Aldrich Cat # 10196-18-6 His-Nus This paper N/A His-Nus-Aurora A-WT, F31I, K162M This paper N/A His-Nus-NEK2 This paper N/A GST This paper N/A GST-OLA1-WT, T124A, T124E, T124E + DM This paper N/A GST-Aurora A-N, DN This paper N/A His-OLA1 This paper N/A Critical commercial assays GenomeLab Human STR Primer set Beckman Coulter Cat # A20100 SilencerTM siRNA construction Kit Thermo Fisher Scientific Cat # AM1620 Experimental models: Cell lines HeLa ATCC Cat # CCL-2; RRID: CVCL_0030 HEK-293T ATCC Cat # CRL-11268 U2OS ATCC Cat # HTB-96 RRID: CVCL_0042 MCF7 ATCC Cat # HTB-22 HeLa-tet-shOLA1 This paper N/A Oligonucleotides siRNA targeting sequence Aurora A-1: 50-AUUCUUCCCAGCGCGUUCC-30 Hirota et al.65 N/A (Continued on next page) Cell Reports 42, 112850, August 29, 2023 21 .. REAGENT or RESOURCE SOURCE IDENTIFIER siRNA targeting sequence Aurora A-2: 50-AUGCCCUGUCUUACUGUCA-30 Kufer et al.66 N/A siRNA targeting sequence Aurora A-3: Integrated DNA Technologies Cat # hs.Ri.AURKA.13.1 Recombinant DNA pCY4B-FLAG-Aurora A This paper N/A pCY4B-FLAG-Aurora A-mutant This paper N/A pCY4B-FLAG-1–133 (N) This paper N/A pCY4B-FLAG-105–403 (DN) This paper N/A pCY4B-HA-Aurora A Yoshino et al.25 N/A pET-Aurora A Yoshino et al.25 N/A pET-Aurora A-mutant This paper N/A pEGFP-Aurora A This paper N/A pEGFP-Aurora A-mutant This paper N/A pEGFP-Aurora A-1–133 (N) This paper N/A pEGFP-Aurora A-105–403 (DN) This paper N/A pGEX-Aurora A-1–133 (N) This paper N/A pGEX-Aurora A-105–403 (DN) This paper N/A pCY4B-HA-OLA1 Matsuzawa et al.16 N/A pCY4B-HA-OLA1-Mutant Yoshino et al.21 This paper N/A pCY4B-HA-OLA1-1–171 (N) This paper N/A pCY4B-HA-OLA1-166–396 (C) This paper N/A pCY4B-FLAG-OLA1 Matsuzawa et al.16 N/A pCY4B-FLAG-OLA1-Mutant This paper N/A pGEX-OLA1 Matsuzawa et al.16 N/A pGEX-OLA1-Mutant Yoshino et al.21 This paper N/A pET-OLA1 Matsuzawa et al.16 N/A pLKO-tet-shOLA1-puro This paper N/A pCMV-Myc-ubiquitin Starita et al.67 N/A pCMV-Myc-ubiquitin-mutant This paper N/A pEGFP-centrin Yoshino et al.21 N/A pEGFP-NEK2 This paper N/A pEGFP-NEK2-K37R This paper N/A pET-NEK2 This paper N/A pCY4B-HA-NEK2 This paper N/A pCY4B-HA-NEK2-K37R This paper N/A psPAX2 Addgene Cat # 12260 pMD2.G Addgene Cat # 12259 EZ-Tet-pLKO-Puro Addgene Cat # 85966 pCY4B-FLAG-mCherry-puro This paper N/A Software and algorithms ImageJ National Institute of Health https://ImageJ.nih.gov/ij/ JMP 12 software JMP https://www.jmp.com/ja_jp/software.html Other ImageQuant LAS 4000mini Cytiva N/A BZ-X710 Keyence N/A 22 Cell Reports 42, 112850, August 29, 2023
Protease Inhibitor:Article Title: Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins MG132 Peptide Institute Cat # 3175-v Puromycin FUJIFILM Wako Cat # 160-23151 Cycloheximide FUJIFILM Wako Cat # 037-20991 Nocodazole FUJIFILM Wako Cat # 140-08531 Doxycycline Sigma-Aldrich Cat #D9891 poly-L-lysine solution Sigma-Aldrich Cat #P8920 MLN8054 AdooQ Bioscience Cat # A10601 Protease inhibitor cocktail Sigma-Aldrich Cat #P8340 Phosphatase inhibitor Cocktail Nacalai Tesque Cat # 07574-61 Polyethylenimine MAX Polysciences Cat # 24765-1 TransIT-X2 Dynamic Delivery System Mirus Bio Cat # MIR6000 Protein G-Sepharose 4 Fast Flow Cytiva Cat # 17-0618-01 Ab-Capcher Protein A-R28 beads ProteNova Cat # P-002-10 anti-DYKDDDDK tag antibody beads FUJIFILM Wako Cat # 012-22781 Glutathione Sepharose 4B beads Cytiva Cat # 17-0756-01 Ni-NTA-agarose Thermo Fisher Scientific Cat #R90115 HA-Peptide Roche Cat # 11-666-975-001 Ubiquitin Sigma-Aldrich Cat #U6253 Recombinant Human Ubiquitin-Activating Enzyme (UBE1) Bio-Techne Cat # E 305-025 Recombinant Human His-UBE2N/MMS2 complex Bio-Techne Cat #E2-666-100 Ubiquitin Conjugation Reaction buffer Kit Bio-Techne Cat # SK-10 Phos-tag Acrylamide FUJIFILM Wako Cat # 304-93521 Acrylamide/Bisacrylamide (37.5:1) FUJIFILM Wako Cat # 018-25625 Wide range gel preparation buffer Nacalai Tesque Cat # 07831-94 Zinc nitrate hexahydrate Sigma-Aldrich Cat # 10196-18-6 His-Nus This paper N/A His-Nus-Aurora A-WT, F31I, K162M This paper N/A His-Nus-NEK2 This paper N/A GST This paper N/A GST-OLA1-WT, T124A, T124E, T124E + DM This paper N/A GST-Aurora A-N, DN This paper N/A His-OLA1 This paper N/A Critical commercial assays GenomeLab Human STR Primer set Beckman Coulter Cat # A20100 SilencerTM siRNA construction Kit Thermo Fisher Scientific Cat # AM1620 Experimental models: Cell lines HeLa ATCC Cat # CCL-2; RRID: CVCL_0030 HEK-293T ATCC Cat # CRL-11268 U2OS ATCC Cat # HTB-96 RRID: CVCL_0042 MCF7 ATCC Cat # HTB-22 HeLa-tet-shOLA1 This paper N/A Oligonucleotides siRNA targeting sequence Aurora A-1: 50-AUUCUUCCCAGCGCGUUCC-30 Hirota et al.65 N/A (Continued on next page) Cell Reports 42, 112850, August 29, 2023 21 .. REAGENT or RESOURCE SOURCE IDENTIFIER siRNA targeting sequence Aurora A-2: 50-AUGCCCUGUCUUACUGUCA-30 Kufer et al.66 N/A siRNA targeting sequence Aurora A-3: Integrated DNA Technologies Cat # hs.Ri.AURKA.13.1 Recombinant DNA pCY4B-FLAG-Aurora A This paper N/A pCY4B-FLAG-Aurora A-mutant This paper N/A pCY4B-FLAG-1–133 (N) This paper N/A pCY4B-FLAG-105–403 (DN) This paper N/A pCY4B-HA-Aurora A Yoshino et al.25 N/A pET-Aurora A Yoshino et al.25 N/A pET-Aurora A-mutant This paper N/A pEGFP-Aurora A This paper N/A pEGFP-Aurora A-mutant This paper N/A pEGFP-Aurora A-1–133 (N) This paper N/A pEGFP-Aurora A-105–403 (DN) This paper N/A pGEX-Aurora A-1–133 (N) This paper N/A pGEX-Aurora A-105–403 (DN) This paper N/A pCY4B-HA-OLA1 Matsuzawa et al.16 N/A pCY4B-HA-OLA1-Mutant Yoshino et al.21 This paper N/A pCY4B-HA-OLA1-1–171 (N) This paper N/A pCY4B-HA-OLA1-166–396 (C) This paper N/A pCY4B-FLAG-OLA1 Matsuzawa et al.16 N/A pCY4B-FLAG-OLA1-Mutant This paper N/A pGEX-OLA1 Matsuzawa et al.16 N/A pGEX-OLA1-Mutant Yoshino et al.21 This paper N/A pET-OLA1 Matsuzawa et al.16 N/A pLKO-tet-shOLA1-puro This paper N/A pCMV-Myc-ubiquitin Starita et al.67 N/A pCMV-Myc-ubiquitin-mutant This paper N/A pEGFP-centrin Yoshino et al.21 N/A pEGFP-NEK2 This paper N/A pEGFP-NEK2-K37R This paper N/A pET-NEK2 This paper N/A pCY4B-HA-NEK2 This paper N/A pCY4B-HA-NEK2-K37R This paper N/A psPAX2 Addgene Cat # 12260 pMD2.G Addgene Cat # 12259 EZ-Tet-pLKO-Puro Addgene Cat # 85966 pCY4B-FLAG-mCherry-puro This paper N/A Software and algorithms ImageJ National Institute of Health https://ImageJ.nih.gov/ij/ JMP 12 software JMP https://www.jmp.com/ja_jp/software.html Other ImageQuant LAS 4000mini Cytiva N/A BZ-X710 Keyence N/A 22 Cell Reports 42, 112850, August 29, 2023
Conjugation Assay:Article Title: Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins MG132 Peptide Institute Cat # 3175-v Puromycin FUJIFILM Wako Cat # 160-23151 Cycloheximide FUJIFILM Wako Cat # 037-20991 Nocodazole FUJIFILM Wako Cat # 140-08531 Doxycycline Sigma-Aldrich Cat #D9891 poly-L-lysine solution Sigma-Aldrich Cat #P8920 MLN8054 AdooQ Bioscience Cat # A10601 Protease inhibitor cocktail Sigma-Aldrich Cat #P8340 Phosphatase inhibitor Cocktail Nacalai Tesque Cat # 07574-61 Polyethylenimine MAX Polysciences Cat # 24765-1 TransIT-X2 Dynamic Delivery System Mirus Bio Cat # MIR6000 Protein G-Sepharose 4 Fast Flow Cytiva Cat # 17-0618-01 Ab-Capcher Protein A-R28 beads ProteNova Cat # P-002-10 anti-DYKDDDDK tag antibody beads FUJIFILM Wako Cat # 012-22781 Glutathione Sepharose 4B beads Cytiva Cat # 17-0756-01 Ni-NTA-agarose Thermo Fisher Scientific Cat #R90115 HA-Peptide Roche Cat # 11-666-975-001 Ubiquitin Sigma-Aldrich Cat #U6253 Recombinant Human Ubiquitin-Activating Enzyme (UBE1) Bio-Techne Cat # E 305-025 Recombinant Human His-UBE2N/MMS2 complex Bio-Techne Cat #E2-666-100 Ubiquitin Conjugation Reaction buffer Kit Bio-Techne Cat # SK-10 Phos-tag Acrylamide FUJIFILM Wako Cat # 304-93521 Acrylamide/Bisacrylamide (37.5:1) FUJIFILM Wako Cat # 018-25625 Wide range gel preparation buffer Nacalai Tesque Cat # 07831-94 Zinc nitrate hexahydrate Sigma-Aldrich Cat # 10196-18-6 His-Nus This paper N/A His-Nus-Aurora A-WT, F31I, K162M This paper N/A His-Nus-NEK2 This paper N/A GST This paper N/A GST-OLA1-WT, T124A, T124E, T124E + DM This paper N/A GST-Aurora A-N, DN This paper N/A His-OLA1 This paper N/A Critical commercial assays GenomeLab Human STR Primer set Beckman Coulter Cat # A20100 SilencerTM siRNA construction Kit Thermo Fisher Scientific Cat # AM1620 Experimental models: Cell lines HeLa ATCC Cat # CCL-2; RRID: CVCL_0030 HEK-293T ATCC Cat # CRL-11268 U2OS ATCC Cat # HTB-96 RRID: CVCL_0042 MCF7 ATCC Cat # HTB-22 HeLa-tet-shOLA1 This paper N/A Oligonucleotides siRNA targeting sequence Aurora A-1: 50-AUUCUUCCCAGCGCGUUCC-30 Hirota et al.65 N/A (Continued on next page) Cell Reports 42, 112850, August 29, 2023 21 .. REAGENT or RESOURCE SOURCE IDENTIFIER siRNA targeting sequence Aurora A-2: 50-AUGCCCUGUCUUACUGUCA-30 Kufer et al.66 N/A siRNA targeting sequence Aurora A-3: Integrated DNA Technologies Cat # hs.Ri.AURKA.13.1 Recombinant DNA pCY4B-FLAG-Aurora A This paper N/A pCY4B-FLAG-Aurora A-mutant This paper N/A pCY4B-FLAG-1–133 (N) This paper N/A pCY4B-FLAG-105–403 (DN) This paper N/A pCY4B-HA-Aurora A Yoshino et al.25 N/A pET-Aurora A Yoshino et al.25 N/A pET-Aurora A-mutant This paper N/A pEGFP-Aurora A This paper N/A pEGFP-Aurora A-mutant This paper N/A pEGFP-Aurora A-1–133 (N) This paper N/A pEGFP-Aurora A-105–403 (DN) This paper N/A pGEX-Aurora A-1–133 (N) This paper N/A pGEX-Aurora A-105–403 (DN) This paper N/A pCY4B-HA-OLA1 Matsuzawa et al.16 N/A pCY4B-HA-OLA1-Mutant Yoshino et al.21 This paper N/A pCY4B-HA-OLA1-1–171 (N) This paper N/A pCY4B-HA-OLA1-166–396 (C) This paper N/A pCY4B-FLAG-OLA1 Matsuzawa et al.16 N/A pCY4B-FLAG-OLA1-Mutant This paper N/A pGEX-OLA1 Matsuzawa et al.16 N/A pGEX-OLA1-Mutant Yoshino et al.21 This paper N/A pET-OLA1 Matsuzawa et al.16 N/A pLKO-tet-shOLA1-puro This paper N/A pCMV-Myc-ubiquitin Starita et al.67 N/A pCMV-Myc-ubiquitin-mutant This paper N/A pEGFP-centrin Yoshino et al.21 N/A pEGFP-NEK2 This paper N/A pEGFP-NEK2-K37R This paper N/A pET-NEK2 This paper N/A pCY4B-HA-NEK2 This paper N/A pCY4B-HA-NEK2-K37R This paper N/A psPAX2 Addgene Cat # 12260 pMD2.G Addgene Cat # 12259 EZ-Tet-pLKO-Puro Addgene Cat # 85966 pCY4B-FLAG-mCherry-puro This paper N/A Software and algorithms ImageJ National Institute of Health https://ImageJ.nih.gov/ij/ JMP 12 software JMP https://www.jmp.com/ja_jp/software.html Other ImageQuant LAS 4000mini Cytiva N/A BZ-X710 Keyence N/A 22 Cell Reports 42, 112850, August 29, 2023
Sequencing:Article Title: Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins MG132 Peptide Institute Cat # 3175-v Puromycin FUJIFILM Wako Cat # 160-23151 Cycloheximide FUJIFILM Wako Cat # 037-20991 Nocodazole FUJIFILM Wako Cat # 140-08531 Doxycycline Sigma-Aldrich Cat #D9891 poly-L-lysine solution Sigma-Aldrich Cat #P8920 MLN8054 AdooQ Bioscience Cat # A10601 Protease inhibitor cocktail Sigma-Aldrich Cat #P8340 Phosphatase inhibitor Cocktail Nacalai Tesque Cat # 07574-61 Polyethylenimine MAX Polysciences Cat # 24765-1 TransIT-X2 Dynamic Delivery System Mirus Bio Cat # MIR6000 Protein G-Sepharose 4 Fast Flow Cytiva Cat # 17-0618-01 Ab-Capcher Protein A-R28 beads ProteNova Cat # P-002-10 anti-DYKDDDDK tag antibody beads FUJIFILM Wako Cat # 012-22781 Glutathione Sepharose 4B beads Cytiva Cat # 17-0756-01 Ni-NTA-agarose Thermo Fisher Scientific Cat #R90115 HA-Peptide Roche Cat # 11-666-975-001 Ubiquitin Sigma-Aldrich Cat #U6253 Recombinant Human Ubiquitin-Activating Enzyme (UBE1) Bio-Techne Cat # E 305-025 Recombinant Human His-UBE2N/MMS2 complex Bio-Techne Cat #E2-666-100 Ubiquitin Conjugation Reaction buffer Kit Bio-Techne Cat # SK-10 Phos-tag Acrylamide FUJIFILM Wako Cat # 304-93521 Acrylamide/Bisacrylamide (37.5:1) FUJIFILM Wako Cat # 018-25625 Wide range gel preparation buffer Nacalai Tesque Cat # 07831-94 Zinc nitrate hexahydrate Sigma-Aldrich Cat # 10196-18-6 His-Nus This paper N/A His-Nus-Aurora A-WT, F31I, K162M This paper N/A His-Nus-NEK2 This paper N/A GST This paper N/A GST-OLA1-WT, T124A, T124E, T124E + DM This paper N/A GST-Aurora A-N, DN This paper N/A His-OLA1 This paper N/A Critical commercial assays GenomeLab Human STR Primer set Beckman Coulter Cat # A20100 SilencerTM siRNA construction Kit Thermo Fisher Scientific Cat # AM1620 Experimental models: Cell lines HeLa ATCC Cat # CCL-2; RRID: CVCL_0030 HEK-293T ATCC Cat # CRL-11268 U2OS ATCC Cat # HTB-96 RRID: CVCL_0042 MCF7 ATCC Cat # HTB-22 HeLa-tet-shOLA1 This paper N/A Oligonucleotides siRNA targeting sequence Aurora A-1: 50-AUUCUUCCCAGCGCGUUCC-30 Hirota et al.65 N/A (Continued on next page) Cell Reports 42, 112850, August 29, 2023 21 .. REAGENT or RESOURCE SOURCE IDENTIFIER siRNA targeting sequence Aurora A-2: 50-AUGCCCUGUCUUACUGUCA-30 Kufer et al.66 N/A siRNA targeting sequence Aurora A-3: Integrated DNA Technologies Cat # hs.Ri.AURKA.13.1 Recombinant DNA pCY4B-FLAG-Aurora A This paper N/A pCY4B-FLAG-Aurora A-mutant This paper N/A pCY4B-FLAG-1–133 (N) This paper N/A pCY4B-FLAG-105–403 (DN) This paper N/A pCY4B-HA-Aurora A Yoshino et al.25 N/A pET-Aurora A Yoshino et al.25 N/A pET-Aurora A-mutant This paper N/A pEGFP-Aurora A This paper N/A pEGFP-Aurora A-mutant This paper N/A pEGFP-Aurora A-1–133 (N) This paper N/A pEGFP-Aurora A-105–403 (DN) This paper N/A pGEX-Aurora A-1–133 (N) This paper N/A pGEX-Aurora A-105–403 (DN) This paper N/A pCY4B-HA-OLA1 Matsuzawa et al.16 N/A pCY4B-HA-OLA1-Mutant Yoshino et al.21 This paper N/A pCY4B-HA-OLA1-1–171 (N) This paper N/A pCY4B-HA-OLA1-166–396 (C) This paper N/A pCY4B-FLAG-OLA1 Matsuzawa et al.16 N/A pCY4B-FLAG-OLA1-Mutant This paper N/A pGEX-OLA1 Matsuzawa et al.16 N/A pGEX-OLA1-Mutant Yoshino et al.21 This paper N/A pET-OLA1 Matsuzawa et al.16 N/A pLKO-tet-shOLA1-puro This paper N/A pCMV-Myc-ubiquitin Starita et al.67 N/A pCMV-Myc-ubiquitin-mutant This paper N/A pEGFP-centrin Yoshino et al.21 N/A pEGFP-NEK2 This paper N/A pEGFP-NEK2-K37R This paper N/A pET-NEK2 This paper N/A pCY4B-HA-NEK2 This paper N/A pCY4B-HA-NEK2-K37R This paper N/A psPAX2 Addgene Cat # 12260 pMD2.G Addgene Cat # 12259 EZ-Tet-pLKO-Puro Addgene Cat # 85966 pCY4B-FLAG-mCherry-puro This paper N/A Software and algorithms ImageJ National Institute of Health https://ImageJ.nih.gov/ij/ JMP 12 software JMP https://www.jmp.com/ja_jp/software.html Other ImageQuant LAS 4000mini Cytiva N/A BZ-X710 Keyence N/A 22 Cell Reports 42, 112850, August 29, 2023
In Vitro:Article Title: The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation
Article Snippet: .. Recombinant proteins required for in vitro ubiquitylation assays were expressed in BL21(DE3) cells, except for UBE1 and the neddylated CUL5–RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025). .. Cells were collected, quickly rinsed with 1× PBS and lysed in lysis buffer (20 mM Tris pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 1.5 mM MgCl 2 , 2 mM EGTA, 2 mM DTT and 1× cOmplete Protease Inhibitor Cocktail (Roche)) on ice for 30 min and centrifuged at 12,000 rpm, 4 °C for 10 min.
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