The SJ26-treated H1299 cells were washed twice with phosphate-buffered saline (PBS) and lysed using radioimmune precipitation assay buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 100 μM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 25 mM dithiothreitol). Equal amount of total proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Antibodies against Wnt1 (1:1000 dilution, Spring, REF E3960), β-catenin (1:1000 dilution, GeneTex, GTX61089), and α-tubulin (1:5000 dilution, Millipore, MAB374) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Biosciences) were used as the secondary antibodies. Blots were visualized using enhanced chemiluminescence system (PerkinElmer Life Sciences).
Enhanced chemiluminescence system
Manufactured by PerkinElmer
Sourced in United States
The Enhanced chemiluminescence system is a lab equipment product that enables the detection and quantification of proteins in Western blot analyses. It utilizes chemiluminescent substrates to produce light signals proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Cellytic m cell lysis reagent, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Protein assay kit, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (2 mentions)
Tween 20, by Fujifilm (2 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Cyclin a2, by Abcam (2 mentions)
Cyclin b1, by Abcam (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Abcam (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Mab374, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse igg or anti rabbit igg, by Cytiva (1 mentions)
X ray film, by PerkinElmer (1 mentions)
Hybond c extra nitrocellulose membrane, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
Anti β actin, by Merck Group (1 mentions)
35 protocols using enhanced chemiluminescence system
1
Wnt1 and β-Catenin Expression Analysis
2
Western Blot Protein Detection Protocol
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Cells were lysed in an ice-cold buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 1% NP-40, 10% glycerol, 1 mM DTT, 1× protease inhibitor cocktail, and 1× phosphatase inhibitor cocktail at 4 °C for 30 min. Cell lysates were separated on a sodium dodecyl sulfate-polyacrylamide gel, and then transferred electrophoretically onto a Hybond-C Extra nitrocellulose membrane (GE Healthcare, Piscataway, NJ, USA). The membrane was pre-hybridized in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween-20 (TBST buffer), and 5% skim milk for 1 h, and then transferred to a solution containing 1% bovine serum albumin/TBST and a primary antibody and incubated overnight at 4 °C. After washing with the TBST buffer, the membrane was submerged in 1% bovine serum albumin/TBST containing an horseradish peroxidase-conjugated secondary antibody for 1 h. The membrane was washed with TBST buffer, and then developed with an enhanced chemiluminescence system (Perkin-Elmer, Boston, MA, USA) and exposed to x-ray film (Roche).
3
Quantitative Proteomic and Transcriptomic Analysis
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The antibodies used in this study were as follows: anti- TERT (MBL, Nagoya, Japan), anti-p16, anti-YB-1 (Abcam, Cambridge, MA, USA), YB-1S102p (Cell signaling Technology, Beverly, Massachusetts, USA), anti-p21-HRP (Santa Cruz, CA, USA), anti-αSMA, anti-β-actin and anti-GAPDH (Sigma, St. Louis). Proteins were visualized using an enhanced chemiluminescence system (Perkin Elmer, Waltham, MA, USA) and scanned for quantitative analysis using an Imager 600 with ImageQuant TL software (GE Healthcare, Chicago, IL, USA).
Taqman primers (Thermo Fisher Scientific) were used for qPCR analysis of human and mouse TERT, αSMA, YB-1, p16, p21, IL6, SIRT1 and 18s rRNA. One-step RT-PCR was performed as before [60 (link)] using a GeneAmp 7500 sequence detection system (Applied Biosystems, Rockford, IL). Results were expressed as 2−ΔΔCT using the indicated control group as calibrator and 18srRNA as reference [61 (link)].
Taqman primers (Thermo Fisher Scientific) were used for qPCR analysis of human and mouse TERT, αSMA, YB-1, p16, p21, IL6, SIRT1 and 18s rRNA. One-step RT-PCR was performed as before [60 (link)] using a GeneAmp 7500 sequence detection system (Applied Biosystems, Rockford, IL). Results were expressed as 2−ΔΔCT using the indicated control group as calibrator and 18srRNA as reference [61 (link)].
4
Western Blot Analysis of Cardiac Proteins
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Proteins were extracted from the heart tissue in a 1x RIPA buffer (Melford, Ipswich, UK). The protein concentration was determined with Bradford assay. Total protein homogenates 20 μg/30 μl were denatured, separated on sodium dodecyl sulfate polyacrylamide electrophoresis gels, and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 2.5 % BSA in Tris- Buffered Saline Tween 20 for 1 h, before being incubated overnight at 4 °C with CD36 (1:1000, Cell Signalling Technology, Cambridge, UK), GLUT-4 (1:100, Santa Cruz, Biotechnology, Heidelberg, Germany), insulin receptor-β (1:1000, Cell Signalling Technology, Cambridge, UK), phosphorylated-PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), AKT, or phosphorylated-AKT (1:500, Abcam, Cambridge, UK). After washing the blots to remove excessive primary antibody binding, the blots were incubated for 1 h with a horseradish peroxydase conjugated secondary antibody at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), housekeeping protein, was used for loading control and protein normalization. The immunoreactive protein bands were developed using the Enhanced Chemiluminescence system (PerkinElmer, Rodgau-Juegesheim, Germany). The intensity of the immunoblot bands was detected with Chemismart 5100.
5
Autophagy Regulation in HCT-8R Cells
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HCT-8R cells were treated with IGF-1, AKT inhibitor and 3-MA. Cells (5×105 cells/ml) were lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The cell extracts were collected and diluted in SDS-loading buffer and denatured for 5 min at 95°C, and protein determination was performed using a bicinchoninic acid assay. The samples (30 µg) were separated using SDS-PAGE on a 12.5% gel and blotted onto 0.2 µm polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, USA). Following blocking with 5% skimmed milk powder in TBS containing Tween 20, membranes were incubated at 4°C overnightwithrabbit antibodies against LC3B (1:400, catalog no. ab48394; Abcam, Cambridge, UK), protein kinase B (1:400, AKT; catalog no. ab179463s; Abcam) and phosphorylated (p)-AKT (1:400, catalog no. ab81283; Abcam). Subsequently, membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody at room temperature for 45 min (1:2,000; catalog no. sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and proteins were visualized with an Enhanced Chemiluminescence system (PerkinElmer, Inc., Waltham, MA, USA). β-actin antibody (1:1,000; catalog no. sc-130656; Santa Cruz Biotechnology, Inc.) was used as an internal control.
6
Western Blot Analysis of Apoptosis Markers
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Cells were washed twice with cold PBS, then total protein was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was quantified by a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). A total of 20 µg protein (for each sample) was loaded and separated by 10% or 12.5% SDS-PAGE, then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C (anti-TGM2, anti-Bcl-2, anti-Bax, anti-akt, anti-p-akt, anti-caspase-3. anti-cleave-caspase-3 and the MAPK family antibody kits; cat. nos. 3557S, 3498, 5023, 8202S, 9926T, 9910T and 4060S; 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA). Following three washes with TBS/0.1% Tween-20 (TBST), the membranes were incubated with anti-rabbit or anti-mouse immunoglobulin G for 1 h at room temperature (cat. nos. 7076 and 7074; 1:5,000 dilution; Cell Signaling Technology, Inc.) and visualized using an enhanced chemiluminescence system (PerkinElmer, Inc., Waltham, MA, USA). Positive immunoreactive bands were densitometrically quantified (Quantity One 1-D, version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to GAPDH.
7
Western Blot Analysis of Bmi1 Protein
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Cells were lysed in lysis buffer CelLytic M Cell Lysis Reagent (Sigma-Aldrich, MA, USA) for 30 min on ice, and the supernatant was collected after centrifugation at 12,000 rpm (Eppendorf 5415D). Cell lysate was separated on a 10% acrylamide gel and transferred to a PVDF membrane. Membranes were blocked in 5% skim milk for 1 h and then incubated with monoclonal antibody against Bmi1 (1:1000, Millipore, USA) or β-actin (1:5000, Millipore, USA) overnight. Membranes were then washed in PBST (PBS with 0.1% Tween-20) 3 times and then incubated for 1 h with secondary antibody. Then the membranes were washed 4 times and developed by an enhanced chemiluminescence system according to the manufacturer's instructions (Perkin Elmer, Waltham, MA).
8
Immunoblotting for Phosphorylated STAT3
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For confirmation of protein phosphorylation, the cytosolic protein extracts and nuclear eluates of the 7-day control and exposure groups were used for Western blotting. The respective cytosolic protein extracts and nuclear eluates from 4 mice in each group were mixed, electrophoresed on polyacrylamide gels, and transferred to polyvinylidene fluoride membrane (GE Healthcare UK Ltd., Little Chalfont, England); the membrane was then blocked with Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (Wako, Osaka, Japan) and 5% skim milk. The membrane was then incubated with a primary rabbit antibody against phosphorylated STAT3 (Tyr705; Cell Signaling Technology, Inc., Boston, MA, USA) and a primary mouse antibody against β-actin (Sigma–Aldrich, St. Louis, MO, USA) for 12 h and washed with Tween-TBS. Next, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) against mouse or rabbit IgG for 1 h, and washed with Tween-TBS. Immunoreactivities to antibodies were visualized using an enhanced chemiluminescence system (PerkinElmer, Inc., Winter Street Waltham, MA, USA).
9
Western Blot Analysis of Protein Targets
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Following the treatments with different drugs, total cellular proteins were obtained using RIPA buffer (Applygen Technologies, Inc., Beijing, China). Protein (30 μg) was added per lane and proteins were separated by SDS-PAGE on 10% gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. Following blocking with 5% skimmed milk at room temperature for 1 h, the membranes were sequentially incubated with primary antibodies overnight at 4 °C. The membrane was washed with TBST (PBS with 0.1% Tween-20) 3 times and then incubated with HRP-conjugated goat (polyclonal) anti-rabbit/anti-mouse IgG secondary antibody for 1 h at room temperature. Then the membrane was washed another 4 times and observed in an enhanced chemiluminescence system (PerkinElmer, Waltham, MA, USA). β-Tubulin was used as a loading control. Antibodies used for western blotting included those against HIF-1α (1:1000, Cell Signaling), P-gp (1:1000, Cell Signaling), connective tissue growth factor (CTGF, 1:1000, Cell Signaling), and Collagen I (1:1000, Cell Signaling).
10
BRAF and CRAF Immunoprecipitation Protocol
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HCC1 and Hep3B cell immunoprecipitation was performed using extracts prepared in lysis buffer [0.5% Nonidet P-40, 100 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl (pH 8.0), containing 1 g/ml leu-peptin, 1 g/ml aprotinin, 1 g/ml pepstatin, 0.5 mM dithio-threitol, 0.2 mM sodium vanadate, 100 nM microcysteine]. The extracts were sonicated on ice and clarified by centrifugation at 10,000 rpm for 15 min. BRAF and CRAF was co-immunoprecipitated using the anti-BRAF monoclonal antibody prebound to Protein G beads for Hep3B. CRAF was co-immunoprecipitated using the anti-BRAF monoclonal antibody (Cell Signaling, Danvers, MA) prebound to sepharose A beads for HCC1 cells. Immobilized immune complexes were washed three times, eluted in sample buffer, resolved by SDS-PAGE (10% gels), and transferred to nitrocellulose for immunoblot analysis. Membranes were blocked in 5% milk diluted in PBS for 1 hr, after which they were incubated with BRAF or CRAF primary antibodies diluted in 5% milk/PBS overnight at 4 °C. Membranes were washed in PBST (PBS with 0.1% Tween-20) three times and then incubated for 1 hr with a secondary antibody. Membranes were washed four times and then developed by an enhanced chemiluminescence system according to the manufacturer’s instructions (PerkinElmer).
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