After removing spent media and washing cells with cold phosphate buffered saline (PBS), the cells were incubated with cold Pierce RIPA lysis buffer (Thermo Scientific, Hudson, NH) containing Halt™ Protease Inhibitor Single-Use Cocktail, Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and 1 mM dithiothreitol (DTT) for 15 min with occasional swirling. Then, the cells were scraped, homogenized with a 26-gauge needle and vortexed at the highest setting for 1 min; the lysates were cleared by centrifuging at 16,000 g at 4°C for 15 min. Protein concentration was determined with the bicinchoninic acid (BCA) method (BCA Protein Assay - Reducing Agent Compatible; Thermo Scientific). Proteins were separated on NuPAGE 4–12% Bis Tris gel electrophoresis (Life Technologies), and transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose; Life Technologies) using iBlot Gel Transfer Device (Life Technologies). The membrane was probed with monoclonal rabbit antibodies, anti-HMGCR (1:500, ab174830, Abcam Inc., Cambridge, MA), anti-vimentin (1:1000, ab92547, Abcam), and anti-E-cadherin (1:1000, 24E10, Cell Signaling Technology, Beverly, MA). A monoclonal mouse antibody to β-actin (1:500, ab8226, Abcam) was used as a loading control. Immunodetection was performed using the iBlot Western Detection chromogenic kit (Life Technologies).
Ab174830
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab174830 is a primary antibody that recognizes human Myc-tag. It is a mouse monoclonal antibody purified from cell culture supernatant.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ab30532, by Abcam (3 mentions)
Ab19862, by Abcam (3 mentions)
Ab8226, by Abcam (2 mentions)
Ab32136, by Abcam (2 mentions)
Ab30682, by Abcam (2 mentions)
Ab134113, by Abcam (2 mentions)
Ab18180, by Abcam (2 mentions)
Ab176323, by Abcam (2 mentions)
Ab65596, by Abcam (2 mentions)
Ab112046, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Enhanced chemiluminescence system, by Merck Group (2 mentions)
Pierce ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel electrophoresis, by Thermo Fisher Scientific (1 mentions)
Bca protein assay reducing agent compatible, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer stacks nitrocellulose, by Thermo Fisher Scientific (1 mentions)
Iblot western detection chromogenic kit, by Thermo Fisher Scientific (1 mentions)
29 protocols using ab174830
1
Analyzing Protein Expression in Cell Lysates
2
Western Blot Analysis of Extracellular Vesicles
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sEVs or cell lysates (10ug) were separated on 7–15% SDS/PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were incubated overnight at 4°C with antibodies specific for: CD34 (1:2000, ab81289, Abcam), CD81 (1:200, PA5-13,582, Thermo Fisher), HMGCR (1:2000, ab174830, Abcam), SREBP-2 (1:500, sc-271,616, Santa Cruz), LDLR (1:500, sc-18,823, Santa Cruz), GAPDH (1:500, FL-335, Santa Cruz), CD123 (1:1000, AF841,R&D), CLL-1 (1:2000, AF2946, R&D), β-Actin (1:500, sc-47,778, Santa Cruz), TSG101 (1:500, PA5-31,260, Thermo Fisher), ApoB (1:2000, 20,578-1-AP, Thermo Fisher), Calnexin (1:1000, #2433, Cell Signalling), Grp94 (1:1000, #20,292, Cell Signalling). Next, the HRP-conjugated secondary antibody (1:10,000, Pierce, Thermo Fisher) was added for 1 h at room temperature (RT) and blots were developed with ECL detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, USA). The intensities of the bands on exposed films were quantified using Image J software (NIH, USA).
3
Western Blot Analysis of Liver Proteins
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A total of 50 mg liver tissues were harvested and the whole proteins were extracted using a mixture of 100 μl lysis buffer, 1 μl protease inhibitor (100× ), 1 μl phosphatase inhibitors (100× ), and 1 μl phenylmethanesulfonyl fluoride (100× ). Protein samples were resuspended in SDS-PAGE sample loading buffer (Beyotime Biotechnology, China) and loaded onto 6 or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) under denaturalizing conditions. After protein separation, it was electro-blotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA), blocked with 5% skim milk powder, followed by overnight immunoblotting at 4°C with diluted primary antibody. The hybridization bands were combined with the corresponding secondary antibodies (Zhongshan, Beijing, China) conjugated with horseradish enzyme at 37°C. Protein concentration was detected via commercially available BeyoECL Star ultra-sensitive ECL chemiluminescence kits (Beyotime Biotechnology, China). Protein expression was quantitated using ImageJ software (NIH, Bethesda, MD, USA). The primary antibodies used in the study were β-actin (1:1,000, TA-09, Zhongshan, Beijing, China), anti-HMGCR (1:1,000, ab174830, Abcam, UK), and anti-SREBP2 (1:500, sc-13552, Santa Cruz Biotechnology, USA).
4
Protein Expression Analysis of Cholesterol Regulators
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Cells plated into 6- or 12-well plates were treated with indicated compounds at varying doses. Whole cell lysates were collected by RIPA Lysis Buffer (Solarbio) with protease inhibitor. The determination of protein concentrations was conducted by BCA assay (Beyotime), Equal cell lysates were electrophoresed through 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes and blotted against different target antibodies at 4 °C overnight. Primary antibodies include Anti-HMGCR (ab174830, Abcam), Anti-Insig-1 (ab112248, Abcam) and Anti-Insig-2 (ab86145, Abcam).
5
Western Blot Analysis of Lipid Metabolism
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Cells grown to near confluence on 6 cm-dishes were lysed, separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Antibodies directed against Abca1 (ab18180), LDL receptor (ab30532), HMG-CoA reductase (ab174830), SREBP-2 active fragment (ab30682), NPC1 (ab134113), and APP (ab32136) were obtained from Abcam (Cambridge, UK). Antibodies to AMPKα (#2603 S) and phospho-Thr172-AMPKα (#2535 S) were from Cell Signaling Technology (Danvers, MA, USA), while anti-β-actin (A5441) was from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). HRP-conjugated secondary antibodies were from GE Healthcare (Freiburg, Germany). The enhanced chemiluminescence system was from Millipore Corporation (Billerica, MA, USA).
6
Western Blot Analysis of Lipid Metabolism Enzymes
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Western blot analysis was carried out as previously described (Corno et al., 2017 (link)). Lysates were fractionated by SDS-PAGE and proteins were blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk and then incubated overnight at 4°C with antibodies to FASN (HPA006461, Sigma-Aldrich, St. Louis, MO, United States), DHCR24 (2033, Cell Signaling Technology, Danvers, MA, United States), stearoyl–CoA desaturase 1 (SCD1) (ab19862 Abcam, Cambridge, UK), mevalonate kinase (MVK) (ab154515, Abcam), acetyl-CoA acetyltransferase 1 (ACAT1) (ab168342, Abcam), ACAT2 (ab131215, Abcam), glutathione peroxidase 4 (GPX4) (sc166120, Santa Cruz Biotechnology, Dallas, Texas, United States), β-Hydroxy β-methylglutaryl-CoA reductase (HMGCR) (ab174830, Abcam). An anti-β-tubulin antibody (ab6046, Abcam) was used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Secondary antibodies were obtained from GE Healthcare.
7
Western Blot Analysis of Lipid Metabolism Proteins
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Protein extraction from cultured cells was performed as described previously [21 (link)]. Samples (30 µg protein/sample) were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Merck). Membranes were blocked for 1 h at room temperature with 5% BSA and incubated overnight at 4 °C with primary antibodies against HMGCR (rabbit anti-HMGCR; ab174830; 1:1,000; Abcam), ACAT-1 (mouse anti-ACAT-1; sc-69,836; 1:1,000; Santa Cruz Biotechnology), LDLR (rabbit anti‐LDLR; ab52818, 1:500; Abcam), and 14-3-3 (rabbit anti-14-3-3, #8312S, 1:1000, Cell Signaling Technology). Membranes were washed, incubated for 1 h with the appropriate secondary antibodies at room temperature, and the immunoblots were developed using enhanced chemiluminescence reagent (ECL plus; Merck). Bands were visualized using the ChemiDoc System. Densitometry of the bands was performed by ImageLab 5.2 version software (Bio-Rad Laboratories, Hercules).
8
Histological Analysis of Murine Osteoarthritis
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Mouse knee joints were harvested and fixed in 10% phosphate‐buffered formalin. Radiographs of the joints were obtained using the Faxitron MX20 X‐ray system. Samples were decalcified in 20% EDTA (pH 8.0), dehydrated, and embedded in paraffin for sectioning, as previously described 4 . Histologic analysis was performed using antibodies against COL10A1 (X53; Quartett) and HMGCR (ab174830; Abcam), and by staining with hematoxylin and eosin and Safranin O. Histomorphometry was performed to measure the osteophyte volume and the bone volume relative to total volume 19 , as previously described 4 .
Grading for OA severity was performed in a blinded manner using the International Cartilage Repair Society (ICRS)20 and Osteoarthritis Research Society International (OARSI) 21 scoring systems, as previously described 4 . For scoring with the ICRS system, images of the knee joints were assessed for 6 features commonly associated with OA, including changes to the subchondral bone, and categories were tallied into an overall score to represent disease severity (lower scores represent greater severity).
Grading for OA severity was performed in a blinded manner using the International Cartilage Repair Society (ICRS)
9
Western Blot Analysis of Liver Proteins
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Frozen mouse liver was homogenized with a Mikro-Dismembrator S (B. Braun Biotech International) in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 20 mM Na4P2O7, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, 200 µM Na3VO4, 40 mM β-glycerolphosphate, 10% glycerol, 1% Triton X-100), separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. SGPL1 was probed with antibody HPA021125 from Atlas Antibodies (Bromma, Sweden). Amyloid precursor protein (ab32136), fatty acid synthase (ab128856), HMG-CoA reductase (ab174830), liver X receptor (ab176323), LDL receptor (ab30532), and NPC-1 (ab134113) antibodies were from Abcam (Cambridge, UK). For PPARγ, the antibody sc-7196 from Santa Cruz Biotechnology (Dallas, TX, USA) was used. Anti-β-actin (#A5441) was from Sigma Aldrich Chemie GmbH. Horseradish peroxidase-conjugated secondary antibodies were from GE Healthcare (Freiburg, Germany), and the enhanced chemiluminescence system was from Merck Millipore (Darmstadt, Germany).
10
Immunoblotting Analysis of Lipid Metabolism
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These procedures were performed as previously described (Bolsoni-Lopes et al., 2013 (link)). Tissues were dissociated using cell lysis buffer containing protease inhibitors (Qiagen, Hilden, Germany). The protein concentrations were measured using the BCA (Thermo Fisher Scientific, USA) method. The tissue protein extracts were separated on SDS-PAGE gels and electro-transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). Specific proteins were detected using the following specific antibodies: beta-actin (1:2,000, CST, 3700S), HMGCR (1:1,000, ABCAm, ab174830), NPC1L1 (1:1,000, ABCAm, ab121000), LXRα (1:1,000, ABCAm, Ab176323), CYP7A1 (ABCAm, ab65596). Secondary horseradish peroxidase-conjugated antibodies were used at 1:2,000 dilutions. The proteins were detected using Western Chemiluminescent HRP Substrate (Cell Signaling Technology, USA). All experiments were repeated at least three times.
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