Cells were lysed with RIPA buffer (Beyotime, Shanghai, China) and quantified with BCA Kit (Beyotime). The same amount of protein was separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and then incubated with the specific antibodies against transcription factor 4 (TCF4; 1:1,000, Invitrogen), β-catenin (1:1,000, Beyotime), SEMA6D (1:1,000, Invitrogen) or GAPDH (1:2,000, Invitrogen) overnight at 4 ℃. Following incubated with secondary antibody (1:2,000, Invitrogen) for 2 h, the membranes were treated with enhanced chemiluminescence solution (Beyotime) to detect the protein signals.
Enhanced chemiluminescence solution
Manufactured by Beyotime
Sourced in China, United States
Enhanced chemiluminescence solution is a laboratory reagent used to detect and quantify protein expression in Western blot analysis. It generates a luminescent signal proportional to the amount of target protein present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (8 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ab6721, by Abcam (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Rabbit polyclonal anti gapdh antibody, by Abcam (2 mentions)
Fluorchem imaging system, by Bio-Techne (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Gapdh, by Bioss Antibodies (2 mentions)
Vimentin, by Bioss Antibodies (2 mentions)
Ab227387, by Abcam (2 mentions)
Ab97051, by Abcam (2 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
Ab6276, by Abcam (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Bca assay, by Beyotime (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
34 protocols using enhanced chemiluminescence solution
1
Western Blot Analysis of Key Signaling Proteins
2
Protein Extraction and Western Blot Analysis
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Extraction of total protein was executed using the RIPA lysis buffer (Beyotime), and western blotting was executed as previously described.25 Antibodies used in the study were as follows: matrix metalloproteinase 2 (MMP2) (ab97779, 1:2000, Abcam), matrix metalloproteinase 9 (MMP9) (ab228402, 1:1000, Abcam), hexokinase 2 (HK2) (ab227198, 1:10000, Abcam), lactate dehydrogenase A (LDHA) (ab125683, 1:1000, Abcam), and NUCKS1 (PA5‐26535, 1:1000, Invitrogen), anti‐β‐actin (ab115777, 1:200, Abcam), and goat anti‐rabbit IgG (ab6721, 1:10000, Abcam). Protein bands were visualized with enhanced chemiluminescence solution (Beyotime).
3
Western Blot Analysis of FGFR1 Protein
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Total protein was extracted from tissue samples and cells with RIPA lysis buffer containing protease inhibitor (Beyotime). Total protein was separated by using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Thereafter, the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime), and the membranes were blocked with tris buffered saline tween buffer with 5% skim milk. The membranes were then incubated with primary antibodies, including anti‐FGFR1 (ab76464, 1:500, Abcam) and anti‐GAPDH (ab9484, 1:1000, Abcam). GAPDH was deemed as a loading control. Next, the membranes were incubated with the secondary antibody (ab6721, 1:5000, Abcam). The immunoblots were visualized with enhanced chemiluminescence solution (Beyotime).
4
Quantitative FGFR1 Protein Analysis
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Western blotting was performed using standard methods. Membranes were probed with polyclonal rabbit antibodies against anti-FGFR1 (1:500; Abcam, Cambridge, MA, U.S.A.). The proteins on membranes were then stripped and re-probed with an anti-GAPDH rabbit polyclonal antibody (Abcam) as a loading control. The blot was developed using enhanced chemiluminescence solution (Beyotime, Haimen, China) and photographed using the FluorChem imaging system (Alpha Innotech Corp., San Leandro, CA, U.S.A.). The intensity of each spot was analyzed with AlphaEaseFC software.
5
MTA2 Protein Expression Analysis
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PTC cells were lysed with radioimmunoprecipitation assay buffer containing protease inhibitor. Equal amounts of protein were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with nonfat milk and incubated with primary antibodies against MTA2 (1:800 dilution; Abcam, Cambridge, MA, USA) or GAPDH (1:10000 dilution; Abcam). After incubation with a secondary antibody (1:2000 dilution; Zhongshan Goldbridge, Beijing, China), they were treated with enhanced chemiluminescence solution (Beyotime Biotech Inc., Jiangsu, China) to detect the protein signals.
6
Western Blot Analysis of Cell Signaling Proteins
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Total protein was extracted from cultured cells using radioimmunoprecipitation assay lysis buffer containing protease inhibitors, and protein concentrations were determined by bicinchoninic acid assay protein assay (Beyotime, Shanghai, China). A total of 20 μg protein lysates was subjected to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were then sealed with 5% skim milk for 2 h, incubated overnight with specific primary antibodies (Table 1 ) and with secondary antibodies for 2 h. Visualization of proteins was performed using the enhanced chemiluminescence solution (Beyotime) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the normalizer.
The Primary Antibodies Used in the Western Blot
| Antibody | Catalog Number | Company | Dilution |
|---|---|---|---|
| NF-κB p65 | Ab207297 | Abcam | 1:2000 |
| phos-NF-κB p65 | Ab222494 | Abcam | 1:2000 |
| Bcl-2 | GTX100064 | Genetex | 1:1000 |
| Bax | #2772 | Cell Signaling Technologies | 1:1000 |
| MDM2 | GTX100531 | Genetex | 1:2000 |
| p53 | GTX70214 | Genetex | 1:5000 |
| GAPDH | Ab8245 | Abcam | 1:10,000 |
7
Western Blot Analysis of Protein Expression
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Total protein was isolated from cells and nude mouse tissues with radioimmunoprecipitation assay lysis buffer (Beyotime). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane and blocked with a 5% nonfat dry milk solution. The membrane was then incubated with one of the following primary antibodies: anti-APC (1:1000, #ab40778, Abcam, UK), anti-phosphorylated-β-catenin (1:500, #9561, Cell Signaling Technology, USA), anti-TET1 (1:3000, #GT1462, Invitrogen, USA) and anti-β-catenin (1:1000, #8480, Cell Signaling Technology, USA). Anti-GAPDH (1:10000, #60004-1-Ig, Proteintech, USA) and Lamin B1 (1:2500, #13435, Cell Signaling Technology, USA) antibodies were used as internal references for total and nuclear proteins, respectively. Lastly, the membrane was incubated with a horseradish peroxidase-conjugated rabbit or mouse secondary antibody, and was developed with an enhanced chemiluminescence solution (Beyotime).
8
Western Blotting Analysis of Liver Proteins
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Western blotting assays were performed as described [38 (link),39 (link)]. The liver and hepatocyte protein were extracted using a protein extraction kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The protein concentration was measured using protein assay reagent (Sangon Biotech). The target proteins were separated in polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a 3% BSA in 0.1% Tris-buffered saline and Tween (TBST) buffer for 4 h. The membranes were hybridized with antibodies specific for PGC-1α (1:1000), Akt (1:1000), GSK-3β (1:1000), P-Akt (1:1000), P-GSK-3β (1:1000), NDUFA9 (1:1000), SDHA (1:1000), UQCRC2 (1:1000), COXIV (1:1000), ATPB (1:1000) (Abcam, USA) and β-Actin (1:5000) (ZSGB-BIO, Beijing, China) overnight at 4 °C. The PVDF membranes were incubated with appropriate peroxidase-conjugated secondary antibodies for 45 min. Immunoreactive bands were detected with enhanced chemiluminescence solution (Beyotime Biotech). The blots were normalized to β-Actin and quantified via densitometry using Image-Pro Plus 6.0 (Media Cybermetics, Rockville, MD, USA). Experiments were carried out in triplicate.
9
Protein Expression Analysis in Liver Tissues
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Liver tissues or hepatocytes were lysed in a cold lysis buffer (Keygene) supplemented with phenylmethanesulfonyl fluoride (PMSF) at 4°C for 30 min. After separation by 12% sulfate‐polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene difluoride membrane. The membrane was blocked at room temperature in phosphate‐buffered saline with Tween 20 buffer with 5% nonfat milk followed by incubation with primary antibody, including anti‐ERO1α (ab172954, Abcam), anti‐GRP78 (ab21685, Abcam), anti‐PERK (ab156919, Abcam), anti‐ATF6 (ab203119, Abcam), and anti‐XBP‐1 (ab37152, Abcam), anti‐DNMT1 (ab13537, Abcam), anti‐G9a (#3306, Cell Signaling Technology), anti‐H3K9me2 (ab176882, Abcam), anti‐H3 (ab1791, Abcam) at 4°C overnight. Blotting of β‐actin (bsm‐33036M, BIOSS) was used as a loading control. The membrane was washed three times and incubated with horseradish peroxidase‐conjugated secondary antibody (ZB‐2306, ZSGB‐BIO) for 2 h. After washing three times, the target proteins were detected using enhanced chemiluminescence solution (Beyotime Institute of Biotechnology), and the relative protein levels were determined by band densitometry using a gel imaging system (Bio‐Rad Laboratories, Inc.).
10
Protein Expression Analysis in Esophageal Cancer
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Total proteins of KYSE30 and KYSE150 cells were extracted using RIPA buffer (Beyotime). The same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocked with nonfat milk, the membranes were incubated with primary antibodies against proliferating cell nuclear antigen (PCNA; 1:1000, Bioss, Beijing, China), Ki-67 (1:200, Bioss), matrix metalloproteinase (MMP2; 1:1000, Bioss), MMP9 (1:500, Bioss), HMGB1 (1:750, Bioss) or GAPDH (1:500, Bioss) at 4°C overnight. Then, the membranes were incubated with secondary antibody (1:1000, Bioss) for 1 h, and the protein signals were visualized using an enhanced chemiluminescence solution (Beyotime).
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