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Protocol for the purification and analysis of nuclear UFMylated proteins.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-10 DOI: 10.1016/j.xpro.2025.103634
Pudchalaluck Panichnantakul, Marlene Oeffinger
{"title":"Protocol for the purification and analysis of nuclear UFMylated proteins.","authors":"Pudchalaluck Panichnantakul, Marlene Oeffinger","doi":"10.1016/j.xpro.2025.103634","DOIUrl":"10.1016/j.xpro.2025.103634","url":null,"abstract":"<p><p>Protein UFMylation regulates numerous cellular processes including ribosome quality control and nuclear DNA repair. Here, we present a technique to isolate nuclei and purify UFMylated proteins under denaturing non-reducing conditions from commonly used mammalian cell line models such as hTERT-RPE1, HEK293, U2OS, and HCT116 cells. We then describe procedures for identifying and analyzing purified UFMylated proteins using mass spectrometry and western blot. For complete details on the use and execution of this protocol, please refer to Panichnantakul et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103634"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11869847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143411079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the in vivo tracing of alveolar type I cells from mice using two distinct dual recombinase-mediated genetic approaches.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-20 DOI: 10.1016/j.xpro.2025.103648
Xueying Yang, Xinfeng Meng, Jingting Zhu, Zhongxiao Wang, Bin Zhou, Kuo Liu
{"title":"Protocol for the in vivo tracing of alveolar type I cells from mice using two distinct dual recombinase-mediated genetic approaches.","authors":"Xueying Yang, Xinfeng Meng, Jingting Zhu, Zhongxiao Wang, Bin Zhou, Kuo Liu","doi":"10.1016/j.xpro.2025.103648","DOIUrl":"10.1016/j.xpro.2025.103648","url":null,"abstract":"<p><p>Revealing the origins of alveolar epithelial stem cells is crucial for preventing and treating lung diseases. Here, we present a protocol for tracing alveolar type I (AT1) cells from mice in vivo using two distinct dual recombinase-mediated systems. We describe steps for model establishment, harvesting tissue, cryosection, immunofluorescence staining, and confocal imaging. We then detail procedures for analysis of image files and quantification. This protocol provides a foundation for elucidating the plasticity of AT1 cell fate during homeostasis and diseases. For complete details on the use and execution of this protocol, please refer to Liu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103648"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11889966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143473043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the purification and crystallization of the Drosophila melanogaster Cfp1PHD domain in complex with an H3K4me3 peptide. 黑腹果蝇 Cfp1PHD 结构域与 H3K4me3 肽复合物的纯化和结晶方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-21 DOI: 10.1016/j.xpro.2025.103649
Sabrina Grégoire, Janelle Grégoire, Monika Joshi, Sabrina Capitani, Sara Chow, Jean-François Couture
{"title":"Protocol for the purification and crystallization of the Drosophila melanogaster Cfp1<sup>PHD</sup> domain in complex with an H3K4me3 peptide.","authors":"Sabrina Grégoire, Janelle Grégoire, Monika Joshi, Sabrina Capitani, Sara Chow, Jean-François Couture","doi":"10.1016/j.xpro.2025.103649","DOIUrl":"10.1016/j.xpro.2025.103649","url":null,"abstract":"<p><p>The tri-methylation of histone H3 on K4 (H3K4me3) is a key epigenetic modification that is predominantly found at active gene promoters and is deposited by the complex of proteins associated with SET1 (COMPASS). CXXC zinc finger protein 1 (Cfp1) regulates this process by recruiting SET1 to chromatin and recognizing H3K4me3 via its plant homeodomain (Cfp1<sup>PHD</sup>). Here, we present a protocol for the purification and crystallization of the Drosophila melanogaster Cfp1<sup>PHD</sup> domain in complex with an H3K4me3 peptide (PDB: 9C0O). We describe steps for obtaining highly pure Cfp1<sup>PHD</sup> and diffraction-quality crystals. We then detail procedures for rapidly identifying crystals containing the H3K4me3-bound form of the Cfp1<sup>PHD</sup> domain. For complete details on the use and execution of this protocol, please refer to Grégoire et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103649"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11904489/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143477097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for performing angiogenic and tumorigenic assays using the in ovo chick embryo chorioallantoic membrane model. 使用卵中鸡胚绒毛膜模型进行血管生成和肿瘤生成试验的规程。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-28 DOI: 10.1016/j.xpro.2025.103663
Tommy Chastel, Serena Filiberti, Stefania Mitola, Roberto Ronca, Andrei Turtoi, Michela Corsini
{"title":"Protocol for performing angiogenic and tumorigenic assays using the in ovo chick embryo chorioallantoic membrane model.","authors":"Tommy Chastel, Serena Filiberti, Stefania Mitola, Roberto Ronca, Andrei Turtoi, Michela Corsini","doi":"10.1016/j.xpro.2025.103663","DOIUrl":"10.1016/j.xpro.2025.103663","url":null,"abstract":"<p><p>The chick embryo chorioallantoic membrane (CAM) is a viable alternative in vivo model to explore cancer biology due to its simplicity, accessibility, and cost-effectiveness. Here, we present a protocol for performing angiogenic and tumorigenic assays using the in ovo chick embryo CAM model. We describe steps for initial general preparation and for the angiogenic and tumorigenic assays in two distinct sections.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103663"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11919581/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538103","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for chromatin immunoprecipitation of chromatin-binding proteins in Schizosaccharomyces pombe using a dual-crosslinking approach. 利用双重交联法对染色质结合蛋白进行染色质免疫沉淀的方法。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-03-13 DOI: 10.1016/j.xpro.2025.103695
Jasbeer S Khanduja, Mo Motamedi
{"title":"Protocol for chromatin immunoprecipitation of chromatin-binding proteins in Schizosaccharomyces pombe using a dual-crosslinking approach.","authors":"Jasbeer S Khanduja, Mo Motamedi","doi":"10.1016/j.xpro.2025.103695","DOIUrl":"10.1016/j.xpro.2025.103695","url":null,"abstract":"<p><p>Single-crosslink chromatin immunoprecipitation (ChIP) is often ineffective at mapping the binding sites of chromatin-binding proteins that indirectly interact with DNA. Here, we present a protocol to map the genomic occupancy of different chromatin regulators and an RNA exosome adapter subunit in Schizosaccharomyces pombe using dual-crosslinking ChIP. We describe steps for cell growth, dual-crosslinking, cell lysis, sonification, and immunoprecipitation. We then detail procedures for washing, crosslink reversal, and DNA purification for downstream analysis using ChIP-qPCR and ChIP sequencing. For complete details on the use and execution of this protocol, please refer to Khanduja et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103695"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11952799/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143630885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for detection of cGAMP-induced STING oligomerization in cultured cells.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 DOI: 10.1016/j.xpro.2025.103709
Lizhi Liu, James L Manley
{"title":"Protocol for detection of cGAMP-induced STING oligomerization in cultured cells.","authors":"Lizhi Liu, James L Manley","doi":"10.1016/j.xpro.2025.103709","DOIUrl":"10.1016/j.xpro.2025.103709","url":null,"abstract":"<p><p>STING (stimulator of interferon genes) plays a critical role in the innate immune response, including in viral infection, cancer immunity, and several autoimmune diseases. Here, we present a protocol for detection of STING oligomerization, a critical step in STING activation. We describe steps for expressing STING in HEK293T cells, activating STING by 2'3' cyclic GMP-AMP (cGAMP) treatment, and analyzing STING oligomerization by non-reducing SDS-PAGE and blue native PAGE. This protocol is useful for studies of STING functions, regulation, and dysregulation. For complete details on the use and execution of this protocol, please refer to Liu and Manley.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103709"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143693626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-24 DOI: 10.1016/j.xpro.2024.103588
Gergő Szanda, Éva Wisniewski, László Barna, Gábor Turu, Ken Mackie
{"title":"A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy.","authors":"Gergő Szanda, Éva Wisniewski, László Barna, Gábor Turu, Ken Mackie","doi":"10.1016/j.xpro.2024.103588","DOIUrl":"10.1016/j.xpro.2024.103588","url":null,"abstract":"<p><p>Microscopic cell segmentation typically requires complex imaging, staining, and computational steps to achieve acceptable consistency. Here, we describe a protocol for the high-fidelity segmentation of the nucleus and cytoplasm in cell culture and apply it to monitor interferon-induced signal transducer and activator of transcription (STAT) signaling. We provide guidelines for sample preparation, image acquisition, and segmentation. The approach performs indistinguishably from neural-network-based segmentation while requiring only conventional and cost-effective techniques. The protocol can be adapted to other signaling molecules undergoing nucleo-cytoplasmic shuttling and to high-throughput applications. This protocol enables simultaneous monitoring of two STAT isoforms using only conventional techniques and equipment and improves upon the assay published in Szanda et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103588"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11969409/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143041610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker and Th1 cytokine detection. 通过活化诱导标记和 Th1 细胞因子检测对人类抗原特异性 T 细胞进行流式细胞术免疫分型的方法。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-19 DOI: 10.1016/j.xpro.2024.103343
Gianluca Rotta, Valentina Achille, Scott J Bornheimer, Melanie Duenas, Marco Fernandez, Arianna Gatti, Sergio Haro Giron, Irene Martinez Rio, Marta Massanella, Diego J Jiménez, Jorge Monserrat, Verena Pfeifer, Josefa Pichler, Barbara Prietl, Harald Sourij, Chiara R M Uras, Sara Vlah, Daniela Fenoglio
{"title":"Protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker and Th1 cytokine detection.","authors":"Gianluca Rotta, Valentina Achille, Scott J Bornheimer, Melanie Duenas, Marco Fernandez, Arianna Gatti, Sergio Haro Giron, Irene Martinez Rio, Marta Massanella, Diego J Jiménez, Jorge Monserrat, Verena Pfeifer, Josefa Pichler, Barbara Prietl, Harald Sourij, Chiara R M Uras, Sara Vlah, Daniela Fenoglio","doi":"10.1016/j.xpro.2024.103343","DOIUrl":"10.1016/j.xpro.2024.103343","url":null,"abstract":"<p><p>Flow cytometry characterization of antigen-specific polyfunctional T cells is a valuable tool to study adaptive immunity. Here, we present a protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker (AIM) and Th1 cytokine detection. We describe steps for preparing peripheral blood mononuclear cells (PBMCs) for stimulation followed by washing and staining PBMCs for flow cytometry. We then detail procedures for acquisition and analysis. This protocol has potential applications in the field of vaccine immunology and immuno-oncology. For complete details on the use and execution of this protocol, please refer to Altosole et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103343"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11728965/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142869709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to assess changes in brain network resistance to perturbation during offline processing using TMS-EEG.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-06 DOI: 10.1016/j.xpro.2025.103622
Martina Bracco, Tuomas P Mutanen, Domenica Veniero, Gregor Thut, Edwin M Robertson
{"title":"Protocol to assess changes in brain network resistance to perturbation during offline processing using TMS-EEG.","authors":"Martina Bracco, Tuomas P Mutanen, Domenica Veniero, Gregor Thut, Edwin M Robertson","doi":"10.1016/j.xpro.2025.103622","DOIUrl":"10.1016/j.xpro.2025.103622","url":null,"abstract":"<p><p>Transcranial magnetic stimulation (TMS) perturbs specific brain regions and, combined with electroencephalography (EEG), enables the assessment of activity within their connected networks. We present a resting-state TMS-EEG protocol, combined with a controlled experimental design, to assess changes in brain network activity during offline processing, following a behavioral task. We describe steps for experimental design planning, setup preparation, data collection, and analysis. This approach minimizes biases inherent to TMS-EEG, ensuring an accurate assessment of changes within the network. For complete details of the use and execution of this protocol, please refer to Bracco et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103622"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851284/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143371236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter. 使用CRISPR-Cas9和一个小基因剪接报告基因产生剪接异构体特异性小鼠突变体的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-04 DOI: 10.1016/j.xpro.2024.103543
Yudong Teng, Kelsey Arbogast, Harald Junge, Zhe Chen
{"title":"Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter.","authors":"Yudong Teng, Kelsey Arbogast, Harald Junge, Zhe Chen","doi":"10.1016/j.xpro.2024.103543","DOIUrl":"10.1016/j.xpro.2024.103543","url":null,"abstract":"<p><p>Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles. For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.<sup>1</sup> and Johnson et al.<sup>2</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103543"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11758566/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142932890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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