B16 cells were cultured in 6-well plates for 48 h, followed by treatment with curcumin (5 µM, 10 µM, 20 µM) for another 24 h. Then, cells were homogenized in RIPA lysis buffer supplemented with protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and 1 mM PMSF. The lysates were incubated on ice for 30 min with a vortexing every 5 min, and centrifuged at 12,000 g for 30 min. The supernatants were harvested, and then the protein concentrations were determined by BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of the proteins (30 µg) was subjected to 10% SDS-PAGE following the immunoblot procedure as demonstrated previously (Lu et al., 2016 (link)). Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 for 90 min, followed by incubation overnight at 4 °C with specific antibodies to Cx32 (1:5,000; ABconel, Wuhan, China), Cx43 (1:5,000; ABconel, Wuhan, China) or GAPDH (1:10,000, ABconel, Wuhan, China). Blots were developed with enhanced chemiluminescence HRP substrate (Millipore, Bedford, MA, USA) and detected by a Tanon detection system (Shanghai, China). The intensities of the blots were quantified with the NIH image J software.
Enhanced chemiluminescence hrp substrate
Manufactured by Merck Group
Sourced in United States
Enhanced chemiluminescence HRP substrate is a laboratory reagent used to detect and quantify the presence of horseradish peroxidase (HRP) in biological samples. It produces a luminescent signal when HRP is present, which can be measured using a luminometer or imaged using a gel documentation system. The substrate is designed to provide a sensitive and stable signal for HRP detection.
Automatically generated - may contain errors
Lab products found in correlation
Gbox chemi xt4 system, by Syngene (2 mentions)
Genetools software, by Syngene (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Detection system, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Immobilon, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Activated caspase 3, by Cell Signaling Technology (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Fgf21, by Santa Cruz Biotechnology (1 mentions)
β klotho, by Abcam (1 mentions)
8 protocols using enhanced chemiluminescence hrp substrate
1
Curcumin Modulates Connexin Expression in B16 Cells
2
Western Blot Analysis of Immune Markers
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Total protein was extracted from mouse brain tissue, and quantitation was performed by direct detect spectroscopy (EMD Millipore). Briefly, protein samples (40 μg) were separated via 10% SDS-PAGE and transferred to Polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA) as previously reported (Ansari et al., 2017). Membranes were incubated with primary antibodies against FOXP3, Helios, GATA3, and IL-17A overnight at 4 °C and subsequently incubated for 2 h with HRP-conjugated secondary antibody (Santa Cruz Biotech, Dallas, TX, USA) at room temperature. The FOXP3, Helios, GATA3, IL-17A and β-actin bands were visualized by enhanced chemiluminescence HRP substrate (Millipore Corporation, Burlington, MA, USA), and their intensity was quantified against the β-actin band used as a loading control [42 (link)].
3
Western Blot Analysis of Fatty Acid Synthase
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Liver (30 mg) was homogenized in lysis cell buffer (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 40 mmol/L β-glycerophosphate, 2 mmol/L Na3VO4, 1 mmol/L PMSF, 1 mmol/L DTT) containing complete protease inhibitor cocktail (Roche Applied Science). Tissue lysates were centrifuged at 1,500 × g for 5 min at 4°C to remove cell debris, and protein content was quantified using the Lowry method. For western blotting, proteins (40 μg) were resolved by SDS-PAGE and transferred to a PVDF membrane (Immobilon; Millipore). Membranes were exposed overnight at 4°C to primary antibodies anti-fatty acid synthase (FAS [C20G5], rabbit monoclonal antibody [mAb] #3180) at 1/1,000 dilution (Cell Signaling, Danvers, MA, USA) and anti-β-actin at 1/1,000 (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) both diluted in 1× PBS containing 0.1% Tween 20, following the recommendations of the manufacturer. After secondary antibody incubation (anti- mouse/rabbit HRP), signals were detected using enhanced chemiluminescence HRP substrate (Millipore) and analyzed with a luminescent image analyzer ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Apoptosis Markers
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Protein concentration was determined using a BCA protein assay kit (CoWin Bioscience, Beijing, China). The protein sample was resolved by 10% denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis and was blotted onto Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA). After being blocked by 5% milk, the membrane was incubated with primary antibodies at 4°C overnight. The antibodies against c-Jun N-terminal kinase (JNK), Phospho-JNK, activated Caspase-3, Bcl-2-associated X (Bax) protein, and Bcl-2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against CYP2E1 was obtained from Abcam (Cambridge, MA, USA), and β-actin was obtained from Huaan Biological Technology (Hangzhou, China). Subsequently, the membrane was incubated in a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody from Thermo Scientific (Rockford, IL, USA) for 1 h. The signal was visualized by enhanced chemiluminescence HRP substrate (Millipore, Billerica, MA, USA) and acquired by GBOX Chemi XT4 System (Syngene, Cambridge, UK). GeneTools software (Syngene) was used for quantification.
5
Western Blot Analysis of Protein Expression
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After the drug treament at corresponding concentration for 24 h, the cells were harvested in lysis buffer (Beyotime Biotechnology) containing protease and phosphatase inhibitors (Roche). Lysates were sonicated for 30 s, and then spun at 12,000 rpm for 10 min at 4°C. Proteins were separated by 6–10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with primary antibodies and horseradish-peroxidase (HRP)-conjugated anti-rabbit IgG antibodies. Target proteins were detected by enhanced chemiluminescence HRP substrate (Millipore).
6
Western Blot Analysis of MAPK, Klotho, and FGF21
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Tissue protein extracts, milk and plasma were analyzed by Western blotting using standard procedures. The utilized primary antibodies were directed against Phospho-p44/42 MAPK (P-Erk1/2) (Thr202/Tyr204) and p44/42 MAPK (Erk1/2) (both from Cell Signaling), β-Klotho (Abcam), FGF21 (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich). Immunoreactive signals were obtained using an enhanced chemiluminescence HRP substrate (Millipore).
7
Protein Extraction and Western Blot Analysis
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Cell pellets were collected after washing with PBS. Total cellular protein was extracted using lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), and the protein concentration was determined with a BCA Kit (Pierce). After boiling in SDS-PAGE loading buffer, 20–40 μg of protein in total cell lysates was fractionated via SDS-PAGE and transferred to PVDF membranes (Millipore). Blots were blocked in 5% BSA (Sigma-Aldrich) and incubated with the appropriate antibodies. The primary antibodies anti-Bcl-10, anti-RelB, anti-p65/Phospho-p65, anti-AKT/Phospho-AKT, anti-ERK/Phospho-ERK, anti-STAT3/Phospho-STAT3 (Cell Signaling), anti-Bcl-xl (Proteintech), anti-GLS1, anti-VDAC1 (Sangon Biotech Shanghai Co., Ltd.), and anti-β-actin (Tianjin Sungene Biotech Co., Ltd.) were used. Goat-anti-mouse IgG/HRP and goat-anti-rabbit IgG/HRP (Cell Signaling) were the secondary antibodies used. The data were acquired using a ChemiDoc™ XRS+ System (Bio-Rad) with enhanced chemiluminescence HRP substrate (Millipore).
8
Immunoblot Analysis of AMPK Signaling
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Proteins were extracted using RIPA lysis buffer, then resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with specific primary antibodies at 4°C overnight. The antibodies against AMPKα, Phosphorylated-AMPKα, Acetyl-CoA Carboxylase (ACC), and Phosphorylated-ACC were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies against SIRT1 and SREBP-1c were obtained from Abcam (Cambridge, MA, USA) and antibodies against suppressor of variegation 39 homolog 2 (SUV39H2), fatty acid synthase (FASN), acyl-Coenzyme A oxidase (ACOX), and peroxisome proliferator activated receptor-α (PPAR-α) from Proteintech (Wuhan, China). Subsequently, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Thermo Scientific, Rockford, IL, USA) at room temperature for 1 h. Next, the enhanced chemiluminescence HRP substrate (Millipore) was added. The signal of protein bands was acquired by GBOX Chemi XT4 System and was finally quantified by Gene Tools software (Syngene, Cambridge, UK).
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