Purification:
Article Title: Identification of Closantel as a small-molecule inhibitor of the compact CRISPR-Cas RNA editor Cas13bt3.
Article Snippet: 1 College of Chemistry, Fuzhou University, Fuzhou 350108, China 2 Department of Emergency, Fujian Provincial Key Laboratory of Emergency Medicine, Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou University Affiliated Provincial Hospital, Fuzhou, China Abstract The type VI CRISPR-Cas systems are widely employed for programmable RNA editing, and the ultra-compact Cas13bt3 ribonuclease offers particular advantages for cellular delivery due to its minimal molecular size.. However, its therapeutic potential is hindered by nonspecific collateral RNA cleavage activity and the lack of small-molecule inhibitors to enable spatiotemporal regulation of its function.. Here, we performed a high-throughput screen for Cas13bt3 inhibitors using a fluorescence resonance energy transfer (FRET)-based RNA cleavage assay.
Cation-Exchange Chromatography:
Transfection:Article Title: Kinase condensates enrich ATP and trigger autophosphorylation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER InvitrogenTM LipofectamineTM 3000 Transfection Reagent Fisher Scientific Cat#L3000008 HaltTM Protease and Phosphatase Inhibitor Cocktail ThermoFisher Scientific Cat#78440 Paraformaldehyde Fisher Scientific Cat#AA433689M TritonTM X-100 Millipore Sigma Cat#X100 TWEEN® 20 Millipore Sigma Cat#P9416 Saponin Millipore Sigma Cat#47036 Benzamidine Millipore Sigma Cat#B6506 Antipain Millipore Sigma Cat#A6191 Leupeptin Millipore Sigma Cat#L2884 Pepstatin Millipore Sigma Cat#P4265 L-Glutathione Millipore Sigma Cat#G4251 Source 15 Q Anion Exchange Resin Cytiva Cat#17094705 Source 15 S Cation Exchange Resin Cytiva Cat17094401 Amylose resin New England Biolabs Cat#E8021L X-tremeGENETM 9 DNA Transfection Reagent Millipore Sigma Cat#6365779001 Critical commercial assays InvitrogenTM ATP Determination Kit Fisher Scientific Cat#A22066 ADP-GloTM Kinase Assay Promega Cat#V6930 GTPase-GloTM Assay Promega Cat#V7681 Micro BCATM Protein Assay Kit ThermoFisher Scientific Cat#23235 Q5® Site-Directed Mutagenesis Kit New England Biolabs Cat#E0554S Deposited data Phospho-MS Data This study Dryad: https://doi.org/10.5061/dryad.j3tx95xwc Experimental models: Cell lines Mouse: MEFs: FAK +/+ ATCC Cat#CRL-2645 Insect: Sf9 Cells Vos laboratory N/A Insect: Sf21 Cells Vos laboratory N/A Insect: HighFiveTM Trichoplusia ni Cells Vos laboratory N/A Recombinant DNA XLone-Puro-EGFP Addgene Cat#140027 XLone-Puro-mEGFP-FAK-WT This study N/A Super PiggyBac Transposase Calo laboratory N/A pFAST-His-TEV-mEGFP-TEV-FAK-WT This study N/A pFAST-His-TEV-mEGFP-TEV-FAK-W266A This study N/A pCMV-mEGFP-FAK-WT Addgene Cat#186148 pCMV-mEGFP-FAK-W266A Addgene Cat#186149 pCMV-mCherry-Paxillin This study N/A pFAST-His-mEGFP-TEV-Abl-WT This study N/A pFAST-His-mEGFP-TEV-Abl-RS This study N/A pCIOX-Mst2 This study N/A pCIOX-mEGFP-Sav1-MBP This study N/A pCIOX-FAK-IDR This study N/A pCIOX-Abl-IDR This study N/A pCIOX-DDX21-C-IDR-WT This study N/A pCIOX-DDX21-C-IDR-RS This study N/A pGEX-PTP1B This study N/A pFAST-His-p130Cas Addgene Cat#186131 pGEX-Paxillin Addgene Cat#186142 pGEX-Nck Addgene Cat#186133 (Continued on next page) 16 Cell Reports ▪▪, 117459, ▪▪, 2026 Article ll OPEN ACCESS .. p53 -/- FAK +/+ mouse embryonic fibroblasts (ATCC CRL-2645) were harvested from E8.0 day old mouse embryo and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 U/mL and 100 mg/mL, respectively) and Glutamax (2 mM) at 37◦C with 5% CO2.
Kinase Assay:Article Title: Kinase condensates enrich ATP and trigger autophosphorylation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER InvitrogenTM LipofectamineTM 3000 Transfection Reagent Fisher Scientific Cat#L3000008 HaltTM Protease and Phosphatase Inhibitor Cocktail ThermoFisher Scientific Cat#78440 Paraformaldehyde Fisher Scientific Cat#AA433689M TritonTM X-100 Millipore Sigma Cat#X100 TWEEN® 20 Millipore Sigma Cat#P9416 Saponin Millipore Sigma Cat#47036 Benzamidine Millipore Sigma Cat#B6506 Antipain Millipore Sigma Cat#A6191 Leupeptin Millipore Sigma Cat#L2884 Pepstatin Millipore Sigma Cat#P4265 L-Glutathione Millipore Sigma Cat#G4251 Source 15 Q Anion Exchange Resin Cytiva Cat#17094705 Source 15 S Cation Exchange Resin Cytiva Cat17094401 Amylose resin New England Biolabs Cat#E8021L X-tremeGENETM 9 DNA Transfection Reagent Millipore Sigma Cat#6365779001 Critical commercial assays InvitrogenTM ATP Determination Kit Fisher Scientific Cat#A22066 ADP-GloTM Kinase Assay Promega Cat#V6930 GTPase-GloTM Assay Promega Cat#V7681 Micro BCATM Protein Assay Kit ThermoFisher Scientific Cat#23235 Q5® Site-Directed Mutagenesis Kit New England Biolabs Cat#E0554S Deposited data Phospho-MS Data This study Dryad: https://doi.org/10.5061/dryad.j3tx95xwc Experimental models: Cell lines Mouse: MEFs: FAK +/+ ATCC Cat#CRL-2645 Insect: Sf9 Cells Vos laboratory N/A Insect: Sf21 Cells Vos laboratory N/A Insect: HighFiveTM Trichoplusia ni Cells Vos laboratory N/A Recombinant DNA XLone-Puro-EGFP Addgene Cat#140027 XLone-Puro-mEGFP-FAK-WT This study N/A Super PiggyBac Transposase Calo laboratory N/A pFAST-His-TEV-mEGFP-TEV-FAK-WT This study N/A pFAST-His-TEV-mEGFP-TEV-FAK-W266A This study N/A pCMV-mEGFP-FAK-WT Addgene Cat#186148 pCMV-mEGFP-FAK-W266A Addgene Cat#186149 pCMV-mCherry-Paxillin This study N/A pFAST-His-mEGFP-TEV-Abl-WT This study N/A pFAST-His-mEGFP-TEV-Abl-RS This study N/A pCIOX-Mst2 This study N/A pCIOX-mEGFP-Sav1-MBP This study N/A pCIOX-FAK-IDR This study N/A pCIOX-Abl-IDR This study N/A pCIOX-DDX21-C-IDR-WT This study N/A pCIOX-DDX21-C-IDR-RS This study N/A pGEX-PTP1B This study N/A pFAST-His-p130Cas Addgene Cat#186131 pGEX-Paxillin Addgene Cat#186142 pGEX-Nck Addgene Cat#186133 (Continued on next page) 16 Cell Reports ▪▪, 117459, ▪▪, 2026 Article ll OPEN ACCESS .. p53 -/- FAK +/+ mouse embryonic fibroblasts (ATCC CRL-2645) were harvested from E8.0 day old mouse embryo and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 U/mL and 100 mg/mL, respectively) and Glutamax (2 mM) at 37◦C with 5% CO2.
Mutagenesis:Article Title: Kinase condensates enrich ATP and trigger autophosphorylation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER InvitrogenTM LipofectamineTM 3000 Transfection Reagent Fisher Scientific Cat#L3000008 HaltTM Protease and Phosphatase Inhibitor Cocktail ThermoFisher Scientific Cat#78440 Paraformaldehyde Fisher Scientific Cat#AA433689M TritonTM X-100 Millipore Sigma Cat#X100 TWEEN® 20 Millipore Sigma Cat#P9416 Saponin Millipore Sigma Cat#47036 Benzamidine Millipore Sigma Cat#B6506 Antipain Millipore Sigma Cat#A6191 Leupeptin Millipore Sigma Cat#L2884 Pepstatin Millipore Sigma Cat#P4265 L-Glutathione Millipore Sigma Cat#G4251 Source 15 Q Anion Exchange Resin Cytiva Cat#17094705 Source 15 S Cation Exchange Resin Cytiva Cat17094401 Amylose resin New England Biolabs Cat#E8021L X-tremeGENETM 9 DNA Transfection Reagent Millipore Sigma Cat#6365779001 Critical commercial assays InvitrogenTM ATP Determination Kit Fisher Scientific Cat#A22066 ADP-GloTM Kinase Assay Promega Cat#V6930 GTPase-GloTM Assay Promega Cat#V7681 Micro BCATM Protein Assay Kit ThermoFisher Scientific Cat#23235 Q5® Site-Directed Mutagenesis Kit New England Biolabs Cat#E0554S Deposited data Phospho-MS Data This study Dryad: https://doi.org/10.5061/dryad.j3tx95xwc Experimental models: Cell lines Mouse: MEFs: FAK +/+ ATCC Cat#CRL-2645 Insect: Sf9 Cells Vos laboratory N/A Insect: Sf21 Cells Vos laboratory N/A Insect: HighFiveTM Trichoplusia ni Cells Vos laboratory N/A Recombinant DNA XLone-Puro-EGFP Addgene Cat#140027 XLone-Puro-mEGFP-FAK-WT This study N/A Super PiggyBac Transposase Calo laboratory N/A pFAST-His-TEV-mEGFP-TEV-FAK-WT This study N/A pFAST-His-TEV-mEGFP-TEV-FAK-W266A This study N/A pCMV-mEGFP-FAK-WT Addgene Cat#186148 pCMV-mEGFP-FAK-W266A Addgene Cat#186149 pCMV-mCherry-Paxillin This study N/A pFAST-His-mEGFP-TEV-Abl-WT This study N/A pFAST-His-mEGFP-TEV-Abl-RS This study N/A pCIOX-Mst2 This study N/A pCIOX-mEGFP-Sav1-MBP This study N/A pCIOX-FAK-IDR This study N/A pCIOX-Abl-IDR This study N/A pCIOX-DDX21-C-IDR-WT This study N/A pCIOX-DDX21-C-IDR-RS This study N/A pGEX-PTP1B This study N/A pFAST-His-p130Cas Addgene Cat#186131 pGEX-Paxillin Addgene Cat#186142 pGEX-Nck Addgene Cat#186133 (Continued on next page) 16 Cell Reports ▪▪, 117459, ▪▪, 2026 Article ll OPEN ACCESS .. p53 -/- FAK +/+ mouse embryonic fibroblasts (ATCC CRL-2645) were harvested from E8.0 day old mouse embryo and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 U/mL and 100 mg/mL, respectively) and Glutamax (2 mM) at 37◦C with 5% CO2.
Recombinant:Article Title: Kinase condensates enrich ATP and trigger autophosphorylation.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER InvitrogenTM LipofectamineTM 3000 Transfection Reagent Fisher Scientific Cat#L3000008 HaltTM Protease and Phosphatase Inhibitor Cocktail ThermoFisher Scientific Cat#78440 Paraformaldehyde Fisher Scientific Cat#AA433689M TritonTM X-100 Millipore Sigma Cat#X100 TWEEN® 20 Millipore Sigma Cat#P9416 Saponin Millipore Sigma Cat#47036 Benzamidine Millipore Sigma Cat#B6506 Antipain Millipore Sigma Cat#A6191 Leupeptin Millipore Sigma Cat#L2884 Pepstatin Millipore Sigma Cat#P4265 L-Glutathione Millipore Sigma Cat#G4251 Source 15 Q Anion Exchange Resin Cytiva Cat#17094705 Source 15 S Cation Exchange Resin Cytiva Cat17094401 Amylose resin New England Biolabs Cat#E8021L X-tremeGENETM 9 DNA Transfection Reagent Millipore Sigma Cat#6365779001 Critical commercial assays InvitrogenTM ATP Determination Kit Fisher Scientific Cat#A22066 ADP-GloTM Kinase Assay Promega Cat#V6930 GTPase-GloTM Assay Promega Cat#V7681 Micro BCATM Protein Assay Kit ThermoFisher Scientific Cat#23235 Q5® Site-Directed Mutagenesis Kit New England Biolabs Cat#E0554S Deposited data Phospho-MS Data This study Dryad: https://doi.org/10.5061/dryad.j3tx95xwc Experimental models: Cell lines Mouse: MEFs: FAK +/+ ATCC Cat#CRL-2645 Insect: Sf9 Cells Vos laboratory N/A Insect: Sf21 Cells Vos laboratory N/A Insect: HighFiveTM Trichoplusia ni Cells Vos laboratory N/A Recombinant DNA XLone-Puro-EGFP Addgene Cat#140027 XLone-Puro-mEGFP-FAK-WT This study N/A Super PiggyBac Transposase Calo laboratory N/A pFAST-His-TEV-mEGFP-TEV-FAK-WT This study N/A pFAST-His-TEV-mEGFP-TEV-FAK-W266A This study N/A pCMV-mEGFP-FAK-WT Addgene Cat#186148 pCMV-mEGFP-FAK-W266A Addgene Cat#186149 pCMV-mCherry-Paxillin This study N/A pFAST-His-mEGFP-TEV-Abl-WT This study N/A pFAST-His-mEGFP-TEV-Abl-RS This study N/A pCIOX-Mst2 This study N/A pCIOX-mEGFP-Sav1-MBP This study N/A pCIOX-FAK-IDR This study N/A pCIOX-Abl-IDR This study N/A pCIOX-DDX21-C-IDR-WT This study N/A pCIOX-DDX21-C-IDR-RS This study N/A pGEX-PTP1B This study N/A pFAST-His-p130Cas Addgene Cat#186131 pGEX-Paxillin Addgene Cat#186142 pGEX-Nck Addgene Cat#186133 (Continued on next page) 16 Cell Reports ▪▪, 117459, ▪▪, 2026 Article ll OPEN ACCESS .. p53 -/- FAK +/+ mouse embryonic fibroblasts (ATCC CRL-2645) were harvested from E8.0 day old mouse embryo and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 U/mL and 100 mg/mL, respectively) and Glutamax (2 mM) at 37◦C with 5% CO2.
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