Transfected cells were solubilized in lysis buffer (150 mM sodium chloride, 0.1 M Tris (pH 8), 1% Tween 20, 50 mM diethyldithiocarbamic acid, and 1 mM ethylenediaminetetra acetic acid (pH 8)) containing protease inhibitors, before being subjected to sonication and centrifugation at 4°C for 3 min. Loading buffer (2×) was added to each of the protein solutions, which were then boiled for 5 min and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to nitrocellulose membranes (Amersham Biosciences, Beijing, China). The membranes were blocked for 1 h with 5% bovine serum albumin in phosphate-buffered saline, incubated with polyclonal antibodies against Ap-2α (1 : 250; Santa Cruz Biotechnology, Beijing, China), and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 2,000; Amersham Pharmacia Biotech). Protein bands were visualized using an enhanced chemiluminescence kit, according to the manufacturer’s specifications (Boster, Wuhan, China).
Enhanced chemiluminescence kit
Manufactured by Boster Bio
Sourced in China
The Enhanced chemiluminescence kit is a laboratory product designed to detect and quantify proteins in Western blot analyses. The kit contains reagents that generate a luminescent signal when interacting with the target proteins, allowing for their visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Abcam (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
Rabbit anti p38, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Rabbit anti gapdh, by Abcam (1 mentions)
Rabbit anti caspase 3, by Abcam (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Boster Bio (1 mentions)
β tubulin, by Boster Bio (1 mentions)
Ultrasensitive s p kit, by Maixin Group (1 mentions)
Ne pertm nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (1 mentions)
Dab reagent, by Maixin Group (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Merck Group (1 mentions)
Protease and phosphatase inhibitors, by MedChemExpress (1 mentions)
5 protocols using enhanced chemiluminescence kit
1
Quantifying Ap-2α in Transfected Cells
2
Western Blot Analysis of Neuroinflammatory Markers
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Cytoplasmic protein extracts were prepared from the left hemispheres (perforation side). The brain tissues were homogenized in an RIPA buffer (Boster Biological Technology, Wuhan, China) and centrifuged (1000×g) at 4°C for 10 minutes. The protein concentration was determined using a BCA protein assay kit (Boster). Equal amounts of total protein (50 μg/sample) were isolated on 10% SDS-polyacrylamide gel and transblotted onto a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk and incubated with specific primary antibodies at 4°C overnight. The primary antibodies used were: rabbit anti-interleukin (IL)-1β (1:1000, Abcam), rabbit anti-tumor necrosis factor (TNF)-α (1:1000, Abcam), rabbit anti-caspase-3 (1:500, Abcam), rabbit anti-P2X4R (1:500, Alomone Labs), rabbit anti-p38 (1:2000, Abcam), rabbit anti-p38 (phosphor T180+Y182, 1:1000, Abcam). GAPDH was detected as a loading control by using rabbit anti-GAPDH (1:5000, Abcam). The next day, membranes were incubated with the secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, Abcam) at room temperature for 1 h after TBST washing. Blot bands were visualized with enhanced chemiluminescence kit (Boster) and band density was quantified by ImageJ software.
3
Western Blot Analysis of Protein Levels
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Liver cancer cell lines and gastric cancer cell lines with a culture density of approximately 80% were washed twice with phosphate-buffered saline (PBS) buffer. Cells were completely lysed with phenylmethylsulfonyl fluoride-spiked high-performance lysis solution (RIPA Lysis Buffer) (Boster, Wuhan), centrifuged (12000 × rpm, 4 ℃, 10 min) and the supernatant collected. Protein concentration was measured using a BCA protein assay kit (Solarbio, Beijing). Each protein sample of 20 μg was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, United States). The membranes were subsequently closed with protein-free fast closure solution (Boster, Wuhan) and rinsed three times for 5 min each time with TBST rinse solution. The membranes were incubated with primary antibodies of PDHB (Proteintech Group, Wuhan) and β-Tubulin (Boster, Wuhan) overnight on a 4 ℃ shaker, followed by incubation with secondary antibodies for 1 h, three rinses with TBST rinse solution for 10 min each time and then with an enhanced chemiluminescence kit (Boster, Wuhan) to visualize the blots. The total grey values of the protein bands were analyzed using ImageJ software to quantify the protein expression levels of the genes.
4
Nuclear and Cytoplasmic Fractionation and Western Blot Analysis
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The NE‐PERTM Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific) was used to isolate and collect cytosolic and nuclear fractions following the manufacturer's protocol. Cells were lysed in radioimmunoprecipitation assay (RIPA) lysate buffer, and cell lysates were incubated on ice for 30 minutes. Cell supernatants were collected, and protein concentrations were determined using bicinchoninic acid (BCA) protein quantitation (Beyotime). SDS‐PAGE electrophoresis was performed on proteins from cell lysate proteins and transferred to PVDF membranes. Membranes were incubated with primary antibody overnight at 4℃. The primary antibodies and secondary antibodies are shown in Table S2 . Proteins in membranes were visualized using an enhanced chemiluminescence kit (BOSTER).
For IHC, mouse tumour sections were dewaxed and rehydrated. The antigen was retrieved under high pressure using citrate buffer (pH = 6.0). The Ultra‐sensitive S‐P kit (Maixin‐Bio) was used to block endogenous peroxidase activity and reduce non‐specific reactivity. Sections were then incubated with primary antibodies (shown in TableS2 ) at 4°C overnight. Mouse tumour sections were then incubated with secondary antibody and streptomycin avidin‐peroxidase using the Ultra‐sensitive S‐P kit, and the sections were visualized with DAB reagent (Maixin‐Bio).
For IHC, mouse tumour sections were dewaxed and rehydrated. The antigen was retrieved under high pressure using citrate buffer (pH = 6.0). The Ultra‐sensitive S‐P kit (Maixin‐Bio) was used to block endogenous peroxidase activity and reduce non‐specific reactivity. Sections were then incubated with primary antibodies (shown in Table
5
Western Blot Analysis of Protein Extraction
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Total protein was extracted using radio immunoprecipitation assay lysis buffer (Millipore, China) containing protease and phosphatase inhibitors (MedChemExpress, USA). The protein concentrations were determined using bicinchoninic acid protein assay. Equal concentrations of proteins were separated using 10% or 12% sodium dodecyl-sulfate polyacrylamide electrophoresis gels (Boster Bio, China) and were transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4 °C with antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The protein bands were incubated in an enhanced chemiluminescence kit (Boster Bio, China) and placed in a gel imaging system for exposure (Beyotime, Shanghai, China). Finally, the densitometric of each band was measured using ImageJ software and normalized to the levels of β-actin.
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