HCC cells were lysed in cell lysis buffer containing 1nM PMSF for 30 min at 4 °C. Lysates were collected by centrifugation at 12,000 rpm for 30 min at 4 °C. Proteins from cell lysates were separated on the SDS-PAGE and transferred onto PVDF membrane (Immobion-P Transfer Membrane, Millipore Corp., Billerica, MA, USA). The membrane was blocked with TBST containing 5% non-fat dry milk for 1 h and further incubated overnight at 4 °C with primary antibodies against PCSK9, LDLR, HMGCR, SREBF2 (Abcam, Cambridge, MA, USA), NF-κB p65, phospho-NF-κB p65, IKKα, TAB3 (Cell Signaling Technology, USA) and β-actin (Santa Cruz, CA, USA). After that, the membrane was incubated with horseradish peroxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnology, USA) for 2 h at room temperature. All protein bands were visualized using an electrochemiluminescence kit (Thermo, USA). Intensity of each protein band was quantified by Quantity One 4.6.2 software (Bio Rad).
Hmgcr
Manufactured by Abcam
Sourced in United States, United Kingdom
HMGCR is a protein that plays a key role in the mevalonate pathway, which is responsible for the production of cholesterol and other important molecules. It catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, a rate-limiting step in this pathway.
Automatically generated - may contain errors
Lab products found in correlation
Srebp2, by Abcam (6 mentions)
Srebf2, by Abcam (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Pcsk9, by Abcam (2 mentions)
Dii ldl, by Thermo Fisher Scientific (2 mentions)
Pcsk9, by Bioneer (2 mentions)
Hmgcr, by Bioneer (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Srebp1, by Abcam (2 mentions)
Abca1, by Abcam (2 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Electrochemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Immobion p transfer membrane, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
20 protocols using hmgcr
1
Western Blot Analysis of Lipid Metabolism Proteins
2
Histological Analysis of Lung Tissues
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Lung tissues from nude mice and clinical samples were fixed at room temperature with 4% paraformaldehyde for ~ 10 h before paraffin embedding. Tissues were then sectioned into 4-μm thick slices. Deparaffinization and rehydration were performed using xylene and a graded series of ethanol, respectively. Hematoxylin and Eosin (H&E) (Beyotime Institute of Biotechnology) was used to stain lung tissues. Clinical samples were incubated with specific primary antibodies against GDF15 (cat.no. ab223539; Abcam), E-cadherin (cat.no. ab40772; Abcam), Vimentin (cat.no. ab92547; Abcam), SCAP (cat.no. ab125186; Abcam), SREBF2 (cat.no. ab30682; Abcam), or HMGCR (cat.no. ab174830; Abcam) at 4°C overnight followed by incubation with the appropriate secondary antibody (cat. no. A0208, Beyotime Institute of Biotechnology) for 30 min at room temperature. DAB was used as the chromogen. Images were captured using a microscope (Leica Microsystems, Germany).
3
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)
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)
4
Protein Expression Analysis in Cancer Cells
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Cancers cells were harvested and extracted with ice-cold RIPA lysis buffer (Solarbio, Beijing, China), supplemented with a protease inhibitor. Proteins were separated by 10% SDS-PAGE, transferred to the PVDF membrane and probed with antibodies against ACLY (Abcam, 1:1000), FASN (Proteintech, Chicago, IL, USA; 1:200), HMGCR (Abcam, 1:1000) and GAPDH (Cell signaling technology, Danvers, MA, USA; 1:3000). A horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Santa Cruz, CA, USA; 1:20000) was incubated and protein expressions were revealed by ECL detection reagents (Millipore, Billerica, MA, USA). The intensity of target protein bands was standardized to the intensity of the GAPDH bands.
5
Immunofluorescence Imaging of HepG2 Cells
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Immunofluorescence of HepG2 cells was performed by following the previously described protocol [17 (link)]. Cells were fixed in paraformaldehyde (4% in PBS) and incubated overnight with appropriate antibodies: HMGCR (Abcam, ab242315, dilution 1:100), SREBP-2 (Abcam, ab30682, dilution 1:100), SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-8984, dilution 1:100), SR-B1 (Abcam, ab52629, dilution 1:100), LDLr (Santa Cruz Biotechnology, sc-11824, dilution 1:200), anti-Perilipin-2 (anti-Plin2) antibody (R&D Systems, #MAB76341, dilution 1:100). After incubation with primary antibodies, fixed cells were probed for 1 hour at room temperature with donkey anti-goat secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, Milan, Italy, A-11055), goat anti-rabbit secondary antibody Alexa Fluor 555 (ThermoFisher Scientific, A27039) and goat anti-rabbit secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, A-11008). Coverslips were mounted with Vectashield Antifade mounting medium with DAPI (Vector, H-1200) to visualize nuclear staining. The samples were examined at confocal microscopy (TCS SP8; Leica, Wetzlar, Germany). Images were captured using Leica TCS SP8 equipped with a 40 × 1.40–0.60 NA HCX Plan Apo oil BL objective at RT and Leica LAS X Software.
6
Investigating Metabolic and Oxidative Stress Responses
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Purified CD4 T cells were treated with 5 μM KML001 or DPBS control for different times and nucleofected cells were harvested and lysed on ice in RIPA lysis buffer (Boston BioProducts Inc, Ashland, MA) in the presence of protease inhibitors (Thermo Fisher Scientific). The protein concentrations were measured by the Pierce BCA protein assay kit (Thermo Fisher Scientific). The proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk, 0.1% Tween-20 in Tris-buffered saline (TBS), and then incubated overnight with primary antibodies for Fasn, PGC1α, NRF1, Pepck, G-6-PD, hmgcs1, ERRα, GPAT, mtTFA, GPx, SOD1, γH2AX, PARP1, TRF2, β-Actin (Cell Signaling, Danvers, MA), PGC1β, HMGCR, DGAT1, PPARα, ACADL, ACADM (Abcam), p53 (Santa Cruz Biotechnology, Dallas, TX), and NEIL1, XRCC1 (Novus Biologicals). Appropriate horseradish peroxide-conjugated secondary antibodies (Cell Signaling) were then used, and the proteins were visualized with the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). The protein bands were captured and quantitatively analyzed by Chemi DocTM MP Imaging System (Bio-Rad).
7
Gamitrinib and LXR623 Receptor Signaling
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Gamitrinib (GTPP) was kindly provided by Dr. Dario Altieri (Wistar Institute, Philadelphia, PA, USA). LXR623 were purchased from Selleckchem (Houston, TX, USA). Low Density Lipoprotein from Human Plasma (LDL) was purchased from Thermo Fisher (LDL-cholesterol). A 10 mM working solution in dimethylsulfoxide (DMSO) was prepared for all reagents prior to storage at –20 °C. Final concentrations of DMSO were below 0.1% (v/v). The following antibodies were used: Mcl-1 (1:500; CST: Cell Signaling Technology, Danvers, MA, USA), BAK (1:500; CST), Bcl-2 (1:500; CST), BIM (1:500; CST), Bcl-xL (1:500; CST), Noxa (1:500, clone 114C307; Calbiochem, Burlington, MA, USA), β-actin (1:000, clone AC15; Sigma Aldrich, St. Louis, MO, USA), ATF4 (1:500; CST), GRP78 (1:500, CST), PERK (1:500, clone D11A8; CST), CHOP (1:500, CST), elF2α (1:25; CST), p-elF2α(1:25; CST), SREBP2 (1:700, Novus Biologicals, Centennial, CO, USA), TRAP1 (1:1000, BD Bioscience, San Jose, CA, USA), HMGCR (1:25, Abcam, Cambridge, MA, USA), Vinculin (1:500; Abcam), PAPRP (1:500; CST), Caspase 9 (1:500; CST), and secondary HRP-linked antibodies were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
8
Sauchinone Modulates Lipid Metabolism
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The HepG2 human hepatocellular liver cell line was obtained from the Korea Research Institute of Bioscience and Biotechnology (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 was purchased from Sigma-Aldrich (St. Louis, MO, USA). DiI-LDL was purchased from invitrogen (Invitrogen, Carlsbad, CA). Antibodies against LSS, SREBP1, SREBP2, PCSK9, HMGCR, LDL-R, SQLE and β-actin were purchased from Abcam, Inc. (Cambridge, MA, USA). PCSK9, LDL-R, HMGCR, SREBP1, SREBP2, HNF1A, BIRC2, ANXA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, South Korea). Sauchinone was isolated from the chloroform fraction of Saururus chinensis, as previously described, and confirmed by a spectroscopic analysis32 (link). The purity of sauchinone was determined to be over 95% by high performance liquid chromatography using an ultraviolet detector.
9
Western Blot Analysis of Lipid Metabolism Proteins
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The samples were lysed using radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using bicinchoninic acid protein assay (G-Biosciences, Maryland Heights, MO, USA). Thirty µg of protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer onto polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). The membranes were blocked at room temperature for one hour using TBS-T (10 mM Tris, 150 mM NaCl, and 0.05% Tween 20, (pH 7.6)) containing 10% skimmed milk, then probed with 1:1000 primary antibodies, such as FAS (Abcam, Cambridge, UK), ACC (Cell signaling, Danvers, MA, USA), SREBP1 (Novus, Centennial, CO, USA), ACOX1, CPT2 (Affinity Biosciences, Pottstown, PA, USA), PPARα (GeneTex, Alton Pkwy Irvine, CA, USA), HMGCR (Abcam, Cambridge, UK) and actin (Abnova, Taipei, Taiwan) at 4 °C overnight. Blots were then washed with TBS-T and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) at room temperature for one hour. Protein bands were visualized using a chemiluminescence horseradish peroxidase substrate (Millipore, Burlington, MA, USA), and the relative signal intensity was quantified using ImageJ software.
10
Western Blot Analysis of Cellular Signaling
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Total proteins were collected from samples using lysis buffer with PMSF and inhibitor cocktail (ThermoFisher, 78443). Protein (20 μg) of each condition was loaded on gradient SDS-PAGE. Samples were separated and transferred onto PVDF membrane. After blocking with 1% BSA (Sigma, A7030), blots were detected by primary antibodies against the following: SREBF2 (28212-1-AP, 1:1000), SQLE (12544-1-AP, 1:2000), PCNA (0205-2-AP, 1:4000), Calnexin (10427-2-AP, 1:4000), p62 (18420-1-AP, 1:2000), Actin (20536-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000) (Proteintech); NPRL2 (37344, 1:2000), TSC2 (4308, 1:1000), phospho-p70S6K (97596, 1:1000), p70S6K (9202, 1:2000) (Cell Signaling); SREBF2 (ab30682, 1:2000), HMGCR (ab242315, 1:1000) (Abcam); and LC3 (Abclonal, A5618, 1:2000).
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