Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay
Sterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regu...
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Bio-protocol LLC
2024-12-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5139&type=0 |
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| author | Mingfeng Xia Tessa Edwards Shunxing Rong |
| author_facet | Mingfeng Xia Tessa Edwards Shunxing Rong |
| author_sort | Mingfeng Xia |
| collection | DOAJ |
| description | Sterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regulate gene expression. Nuclear SREBP levels can be determined by immunoblot analysis; however, this method can only determine the steady-state levels of nuclear SREBPs and does not capture the actual status of activation. The vesicle budding assay provides an alternative way to quantify the activation of SREBPs by monitoring the initiation of SREBP translocation from the ER to the Golgi through vesicles. Microsomal membranes isolated from the liver are incubated in a reaction buffer containing the necessary components to facilitate vesicle formation. Microsomal membranes and vesicles are isolated and SREBPs are quantified in each by immunoblot analysis. The amount of SREBPs found in the budded vesicles provides an assessment of the SREBP activation in the liver. |
| format | Article |
| id | doaj-art-b41efda12bff4c3da0eeebd7e371b460 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-b41efda12bff4c3da0eeebd7e371b4602025-02-07T08:16:22ZengBio-protocol LLCBio-Protocol2331-83252024-12-01142410.21769/BioProtoc.5139Assessment of SREBP Activation Using a Microsomal Vesicle Budding AssayMingfeng Xia0Tessa Edwards1Shunxing Rong2Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USADepartment of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, ChinaCenter for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USACenter for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX, USADepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USASterol regulatory element binding proteins (SREBPs) are transcription factors that reside in the endoplasmic reticulum (ER) membrane as inactive precursors. To be active, SREBPs are translocated to the Golgi where the transcriptionally active N-terminus is cleaved and released to the nucleus to regulate gene expression. Nuclear SREBP levels can be determined by immunoblot analysis; however, this method can only determine the steady-state levels of nuclear SREBPs and does not capture the actual status of activation. The vesicle budding assay provides an alternative way to quantify the activation of SREBPs by monitoring the initiation of SREBP translocation from the ER to the Golgi through vesicles. Microsomal membranes isolated from the liver are incubated in a reaction buffer containing the necessary components to facilitate vesicle formation. Microsomal membranes and vesicles are isolated and SREBPs are quantified in each by immunoblot analysis. The amount of SREBPs found in the budded vesicles provides an assessment of the SREBP activation in the liver.https://bio-protocol.org/en/bpdetail?id=5139&type=0 |
| spellingShingle | Mingfeng Xia Tessa Edwards Shunxing Rong Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay Bio-Protocol |
| title | Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay |
| title_full | Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay |
| title_fullStr | Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay |
| title_full_unstemmed | Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay |
| title_short | Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay |
| title_sort | assessment of srebp activation using a microsomal vesicle budding assay |
| url | https://bio-protocol.org/en/bpdetail?id=5139&type=0 |
| work_keys_str_mv | AT mingfengxia assessmentofsrebpactivationusingamicrosomalvesiclebuddingassay AT tessaedwards assessmentofsrebpactivationusingamicrosomalvesiclebuddingassay AT shunxingrong assessmentofsrebpactivationusingamicrosomalvesiclebuddingassay |