Srebp2

Manufactured by Abcam

Sourced in United States, United Kingdom

SREBP2 is a transcription factor that regulates the expression of genes involved in cholesterol biosynthesis and uptake. It is a member of the sterol regulatory element-binding protein (SREBP) family of transcription factors.

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Lab products found in correlation

Hmgcr, by Abcam (6 mentions) Srebp1, by Santa Cruz Biotechnology (6 mentions) β actin, by Santa Cruz Biotechnology (4 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Srebp1, by Abcam (3 mentions) Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions) Pcsk9, by Bioneer (2 mentions) Hmgcr, by Bioneer (2 mentions) β actin, by Abcam (2 mentions) Fibronectin, by Merck Group (2 mentions) Phosphatase inhibitor cocktail, by Merck Group (2 mentions) Pai 1, by Innovative Research (2 mentions) α tubulin, by GenScript (2 mentions) Luminata forte, by Merck Group (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Type 1 collagen, by Southern Biotech (2 mentions) Cyp7a1, by Abcam (2 mentions) Cyp7b1, by Proteintech (2 mentions)

Close spelling variants of this product

Anti-SREBP2 antibody (7 citations) Anti-SREBP2 (17 citations) Anti-SREBP2 (ab30682; (2 citations)

29 protocols using srebp2

HepG2 Cell Culture and Characterization

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The HepG2 human
hepatocellular liver cell line was obtained from the Korea Research
Institute of Bioscience and Biotechnology (Daejeon, South Korea) and
grown in Eagle’s minimum essential medium (EMEM) containing
10% fetal bovine serum and 100 U/mL penicillin/streptomycin sulfate.
Cells were incubated in a humidified 5% CO₂ atmosphere
at 37 °C. EMEM, penicillin, and streptomycin were purchased from
Hyclone (Logan, UT, USA). Bovine serum albumin (BSA) and 25-hydroxycholesterol
were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4′,6-Diamidino-2-phe-nylindole
(DAPI) and DiI-LDL were purchased from Thermo Fisher Scientific (Waltham,
MA, USA). Antibodies against LSS, SREBP1, SREBP2, HMGCR, SQLE, and
β-actin were purchased from Abcam, Inc. (Cambridge, MA, USA).
Antibodies against PCSK9 were purchased from Cell Signaling Technology.
(Beverly, MA, USA). SREBF1, SREBF2, HMGCR, PCSK9, IDI1, SQLE, ABCA1,
ACSL6, DHCR7, FDFT1, FDPS, SOAT1, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) oligonucleotide primers were purchased from Bioneer Corp.
(Daejeon, South Korea). α-Mangostin was isolated from the chloroform
fraction of G. mangostana, as previously
described, and confirmed by a spectroscopic analysis. The purity of
α-mangostin was determined to be over 95% by high-performance
liquid chromatography using an ultraviolet detector.^{4 (link)}

Chae H.S., Kim H.J., Ko H.J., Lee C.H., Choi Y.H, & Chin Y.W. (2020). Transcriptome Analysis Illuminates a Hub Role of SREBP2 in Cholesterol Metabolism by α-Mangostin. ACS Omega, 5(48), 31126-31136.

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Western Blot Analysis of Lipid Metabolism

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After harvesting, the cells were lysed in protein extraction solution (Intron, Daejeon, Korea). Equal amounts of protein were then loaded and separated on a sodium dodecyl sulfate-polyacrylamide gels, and then the proteins were transferred onto nitrocellulose membranes (Pall Corp., Port Washington, NY, USA). After blocking with 5% skim milk, the membranes were incubated with various primary antibodies. The blots were then incubated with peroxidase-conjugated secondary antibody, and enhanced chemiluminescence (Biomax, Seoul, Korea) was used to visualize the specific proteins. The primary antibodies used in western blot analysis were as follows: actin, sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SREBP-2, farnesyl-diphosphate farnesyltransferase-1 (FDFT-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, mitochondrial transcription factor A (mtTFA), p62, Parkin (Abcam, Cambridge, UK), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-γ (PPAR-γ), insulin-like growth factor-1 receptor (IGF-1R), phospho-IGF-1R, Akt, phospho-Akt, mechanistic target of rapamycin (mTOR), phospho-mTOR, (Cell Signalling Technology, Danvers, MA, USA), stearoyl-coenzyme A desaturase (SCD) (Thermo Scientific, Rockford, IL, USA), and LC-3 (Sigma, St. Louis, MO, USA).

Yoo J.G., Li X.M., Lee J.K., Park S., Hong D., Jung K.E., Lee Y., Seo Y.J., Kim C.D., Shin J.M, & Choi C.W. (2021). Azidothymidine Downregulates Insulin-Like Growth Factor-1 Induced Lipogenesis by Suppressing Mitochondrial Biogenesis and Mitophagy in Immortalized Human Sebocytes. Annals of Dermatology, 33(5), 425-431.

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Immunofluorescence Localization of Lipid Regulators

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HepG2 cells were fixed by 4% paraformaldehyde for 10 minutes at room temperature after being washed by PBS twice. Then cells were blocked and permeabilized with 0.1% Triton X-100 (MP Biomedicals) and 10% FBS diluted in PBS for 30 minutes at room temperature. After that, cells were incubated with primary antibody SREBP1 (Thermo Fisher Scientific, 1:100), SREBP2 (Abcam, 1:100), INSIG1 (Proteintech, 1:200), CALNEXIN (Enzo, 1:200), and PDI (MilliporeSigma, 1:200) incubated at 4°C overnight. After 5 washes with PBS, cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 568 goat anti-mouse IgG (H+L) secondary antibody (Life Technologies) for 1 hour at room temperature with gentle shaking. At last, the samples were washed with PBS 3 times before staining the nucleus with DAPI for 5 minutes. Immunofluorescence images were obtained and analyzed with Zeiss 710 NLO confocal microscopy.
For fluorescence intensity quantification, ImageJ was applied, and relative intensity was quantified by intensity divided by view area. For colocalization, the rate was quantified using ImageJ with colocalization finder. For each sample, no less than 5 representative images were analyzed to quantify the fluorescence intensity values and calculate an average. Experiments were repeated 3 times individually at least.

Xu Y., Tao J., Yu X., Wu Y., Chen Y., You K., Zhang J., Getachew A., Pan T., Zhuang Y., Yuan F., Yang F., Lin X, & Li Y.X. (2021). Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression. JCI Insight, 6(19), e147038.

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Molecular Mechanisms of Irisin Regulation

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Antibodies used in the study were: pAMPKα Thr172 (2535, CST, Beverly, MA, USA) and AMPKα (2532, CST), SREBP2 (ab30682, Abcam, Cambridge, MA, USA) and β-actin (AT 0001, Milwaukee, WI). Donkey-anti-rabbit Alexa Fluor® 488-IgG (711-545-152) was from Jackson ImmunoResearch (West Grove, PA, USA). IRDye-conjugated affinity purified anti-rabbit and anti-mouse IgGs were purchased from Rockland (Gilbertsville, PA, USA). Irisin (067-16) was from Pheonix (Burlingame, CA, USA). Recombinant irisin-Fc and Fc control were expressed in HEK293 cells (Abgent, Nanjing, China) and purified by high-performance liquid chromatography. Oleic acid (OA), collagenase IV and compound C were purchased from Sigma Aldrich (St. Louis, MO, USA). Alzet microosmotic pumps (1002) were from DURECT Corporation (Cupertino, CA, USA). Aprotinin was purchased from Amersham Biosciences (Pittsburgh, PA, USA). Triglyceride and cholesterol Colorimetric Assay Kits were from Cayman Chemical Company (Ann Arbor, MI, USA). BCA protein quantitative assay kit was from Applied Gene (Beijing, China).

Tang H., Yu R., Liu S., Huwatibieke B., Li Z, & Zhang W. (2016). Irisin Inhibits Hepatic Cholesterol Synthesis via AMPK-SREBP2 Signaling. EBioMedicine, 6, 139-148.

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Immunofluorescent Quantification of SREBP2

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Cells were fixed in 4% paraformaldehyde (pH 7.4) for 10 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. Nonspecific binding was blocked with Blocking One HIST (Nacalai, Kyoto, Japan) for 10 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, and then labeled with appropriate fluorescent-tagged secondary antibodies. DAPI (Thermo Fisher Scientific) was used to label nuclei. Acquisition of fluorescence images and quantification were performed using In Cell Analyzer 6500 (GE Healthcare). The following primary antibodies were used in this assay: SREBP2 (Abcam, Emeryville, CA, 1:50).

Egawa N., Izumi Y., Suzuki H., Tsuge I., Fujita K., Shimano H., Izumikawa K., Takahashi N., Tsukita K., Enami T., Nakamura M., Watanabe A., Naitoh M., Suzuki S., Seki T., Kobayashi K., Toda T., Kaji R., Takahashi R, & Inoue H. (2022). TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2. Scientific Reports, 12, 7988.

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Evaluating PAI-1 Inhibitor Effects on Extracellular Matrix

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At the end of treatment with PM_2.5 in the absence and presence of PAI-1 inhibitor TM5614, supernatants from control and treated wells were collected. The cell lysates were prepared using RIPA lysis buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher Scientific) containing protease inhibitor cocktail (cOmplete) and phosphatase inhibitor cocktail (PhosSTOP) (Sigma). Cell lysates were pooled from 3 wells for each control and treatment group and equal amounts of protein were subjected to gel electrophoresis, transferred to PVDF membranes, and subjected to western blotting using antibodies against PAI-1 (Molecular Innovations, Inc.), type I collagen (Southern Biotech), fibronectin (Millipore), sterol regulatory element binding protein-1 and 2 (SREBP1, SREBP2), nuclear factor erythroid related factor 2 (Nrf2) (Abcam), and α-tubulin (GenScript), with HRP-tagged corresponding secondary antibodies. The membranes were developed with enhanced chemiluminescence reagents (Luminata Forte, Millipore, Billerica, MA), and images of protein bands were captured using a BIO-RAD ChemiDoc XRS system (BIO-RAD, CA).

Ghosh A.K., Soberanes S., Lux E., Shang M., Aillon R.P., Eren M., Budinger G.R., Miyata T, & Vaughan D.E. (2021). Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5. Environmental pollution (Barking, Essex : 1987), 287, 117283.

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Western Blot Profiling of Cholesterol Homeostasis

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Equal amounts of protein were loaded onto pre-cast midi-gels (4–12% Bis-Tris; Invitrogen), separated by electrophoresis and transferred onto nitrocellulose membranes using the iBlot™ 2 gel transfer device (Thermo). Membranes were blocked in 5% non-fat milk or 5% bovine serum albumin (BSA) in Tris-buffered saline, 0.1% Tween-20 (TBST) for 1h before incubation with primary antibodies. Primary antibodies included: ABCa1 (1:500, Abcam), apoE (1:1000), LXRβ (1:1000), CYP46 (1:1,000), LDLR (1:500), SREBP2 (1:1000), HMCGR (1:1000), PSD95 (1:1000) (all from Abcam) and loading control GAPDH (1:1000; Santa Cruz). Membranes were incubated with primary antibodies overnight at 4°C, washed in 1X TBST, incubated with appropriate secondary anti-mouse or -rabbit antibodies (1:10,000; Thermo Scientific) for 1h, and developed with ECL Prime (Amersham Pharmacia Biotech, Piscataway, NJ). Band intensities were calculated using ImageJ software and normalized to the loading control (Rasband 1997 ).

Cotto B., Natarajaseenivasan K., Ferrero K., Wesley L., Sayre M, & Langford D. (2018). Cocaine and HIV-1 Tat Disrupt Cholesterol Homeostasis in Astrocytes: Implications for HIV-Associated Neurocognitive Disorders in Cocaine User Patients. Glia, 66(4), 889-902.

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Quantification of Intestinal Lipid Regulators

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The small intestine tissue samples were lysed in immunoprecipitation lysis buffer (Beyotime, Nantong, China). The lysates were homogenized and centrifuged. The supernatants were collected and the protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime). Equal amounts (30 μg) of ABCA1, ATP-binding cassette G1 (ABCG1), ATP-binding cassette G8 (ABCG8), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), low-density lipoproteins receptor (LDLR), LXRα, NPC1L1, PPARα, Sar1B GTPase (Sar1B), scavenger receptor B (SR-B1), SREBP-1, and SREBP-2 were determined by Western blot analysis. Further, the antibodies of ABCA1, ABCG1, ABCG8, ACC, FAS, LDLR, LXRα, NPC1L1, PPARα, Sar1B, SR-B1, SREBP-1, and SREBP-2 were purchased from Abcam (Cambridge, MA, U.S.A.), Cell Signaling Technology (Danvers, MA, U.S.A.), EMD Millipore (Billerica, MA, U.S.A.), or Thermo Fisher Scientific, Inc (Waltham, MA, U.S.A.). Antibody reactivity was detected by Chemiluminescence ECL Detection Systems (EMD Millipore, Billerica, MA, U.S.A.). Subsequently, the intensity of the bands was quantified by densitometry via Gene Tool according to the manufacturer’s instructions (SynGene, Chemi Genius2, PerkinElmer, Wesville, U.S.A.). Beta-actin was used as internal control.

Han S., Zhang W., Zhang R., Jiao J., Fu C., Tong X., Zhang W, & Qin L. (2019). Cereal fiber improves blood cholesterol profiles and modulates intestinal cholesterol metabolism in C57BL/6 mice fed a high-fat, high-cholesterol diet. Food & Nutrition Research, 63, 10.29219/fnr.v63.1591.

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Rat PCSK9 ELISA Assay Protocol

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CircuLex rat PCSK9 enzyme-linked immunosorbent assay (ELISA) kit was purchased from CycLex Co., Nagano, Japan. The antibodies of PCSK9, LDLR and SREBP2 were purchased from Abcam Inc., Cambridge, MA, USA. The anti-hepatocyte nuclear factor-1 alpha (HNF-1α) was obtained from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signal Technology Inc., Beverly, MA, USA.

Xu R.X., Liu J., Li X.L., Li S., Zhang Y., Jia Y.J., Sun J, & Li J.J. (2015). Impacts of ezetimibe on PCSK9 in rats: study on the expression in different organs and the potential mechanisms. Journal of Translational Medicine, 13, 87.

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Western Blot Analysis of SREBP and Inflammatory Markers

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Cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail. Protein extracts were separated on 8%–10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked by incubation for 1 h with 5% non-fat milk, and blotted overnight with the specific antibodies at 4 °C followed by the corresponding secondary antibodies. Then, signals were detected with Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Band densitometry was performed using by ImageJ 1.46r software (National Institute of Health, USA), and relative protein expression was determined by normalizing to β-actin. The following antibodies were used: SREBP-2 (#ab30682, Abcam, USA); SREBP-1 (#sc-8984) and β-actin (#sc-81178) (Santa Cruz Biotechnology, USA); TNF-α (#A0277) (Abclonal, China); anti-rabbit or mouse secondary antibodies (ZSGB-BIO, Beijing, China).

Xiao P.T., Kuang Y.J., Liu S.Y., Xie Z.S., Hao J.H, & Liu E.H. (2022). The antihyperlipidemic equivalent combinatorial components from peel of Citrus reticulata ‘Chachi’. Journal of Food and Drug Analysis, 30(1), 77-87.

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