The BMDMs at 2 × 106 cells per well density in 6-well plates were pretreated with CGP57380 for 15 min, then stimulated with 100 ng/ml LPS for 1 h. The cells were homogenized in radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China) supplemented with phenylmethanesulfonyl fluoride (Beyotime, Jiangsu, China). A bicinchoninic acid extraction kit (Wuhan Boster Biological Technology, Ltd.) was used to determine the protein concentration. The 20 μg denatured protein samples were separated on SDS-PAGE followed by transfer to the nitrocellulose membrane. After blocking, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were against eIF4E, phospho-eIF4E, phospho-ERK, phospho-JNK, phospho-p38 MAPK, and GAPDH. After washing, the membranes were incubated for 2 h with the secondary antibody at room temperature. The bands were visualized using enhanced chemiluminescence (ECL) stain kit and then auto-radiographed.
Radioimmunoprecipitation assay lysis buffer
Manufactured by Wuhan Boster Biological Technology
Sourced in China
Radioimmunoprecipitation assay lysis buffer is a solution used to extract and solubilize proteins from cells or tissues for analysis. It contains a mixture of detergents, salts, and buffering agents that disrupt cell membranes and release intracellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ecl chemiluminescence kit, by Merck Group (2 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Wuhan Boster Biological Technology (1 mentions)
β actin, by Media Cybernetics (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Wnt3a, by Abcam (1 mentions)
Glycogen synthase kinase 3β, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Wuhan Boster Biological Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
β catenin, by Abcam (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab189403, by Abcam (1 mentions)
Ab31392, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bca protein quantitative kit, by Boster Bio (1 mentions)
5 protocols using radioimmunoprecipitation assay lysis buffer
1
Western Blot Analysis of LPS-Induced Signaling
2
Western Blot Analysis of NF-κBp65 in NP Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were prepared from NP tissues using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was measured using a bicinchoninic acid Protein assay kit (Wuhan Boster Biological Technology, Ltd.). Protein samples (20 µg) were then separated using 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membrane was subsequently blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with primary anti-nuclear factor (NF)-κBp65 (cat. no. 8242) and β-actin (cat. no. 4970) antibodies (Cell Signaling Technology, Inc.) at a dilution of 1:1,000 overnight at 4°C. Samples were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore). Protein levels were calculated relative to β-actin and Image-ProPlus software (version 6.0; Media Cybernetics, Inc.) was used for densitometry analysis.
3
Western Blot Analysis of MSCs Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MSCs from three normal healthy subjects were randomly selected and seeded into 6-well plates at 1×105 cells/well after four culture passages. Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was quantified using a BCA Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). The protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Following blocking with 5% skimmed milk powder at 37°C for 1 h, the membranes were incubated with primary antibodies against Smad9 (cat. no. ab115900; 1:500; Abcam), Wnt3a (cat. no. ab28472; 1:500; Abcam), β-catenin (cat. no. 8480; 1:1,000; Cell Signaling Technology, Inc.), glycogen synthase kinase (GSK)-3β (cat. no. 12456; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight, followed by a HRP-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1054; 1:5,000; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The protein bands were visualized using a ChemiDoc™ MP imaging system (Bio-Rad Laboratories, Inc.). Protein levels were calculated relative to β-actin.
4
Breast Tissue Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples were drawn from breast tissues of rats by using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd). Protein was quantified with a BCA protein quantitative kit (Boster). The protein samples (20 μg) were separated by 10% SDS-PAGE and then transferred to a PVDF (Millipore, USA) membrane. Following blocking with 5% BSA, the membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies included rabbit anti-PTEN (1 : 1,000; ab31392; Abcam, CA, USA), mouse anti-PI3K p85 (1 : 1,000; ab189403; Abcam), rabbit anti-p-Akt (1 : 500; ab38449; Abcam), and mouse anti-β-actin (1 : 1,000; ab8226; Abcam). Then, the membranes were rinsed with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (ab6721; 1 : 5,000; Abcam) or HRP-conjugated goat anti-mouse IgG (ab205719; 1 : 5,000; Abcam) at room temperature for 2 h. β-Actin was used as an inner loading control. The protein bands were identified by an ECL chemiluminescence kit (Millipore) and analyzed by Image-Pro Plus software.
5
EGFR Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 1×103 cells/well in 3-well plates for 48 h and washed for 5 min three times in ice-cold PBS. Protein was extracted using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China). Total protein (20 µg/lane) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA), followed by incubation with 10 ml 5% skim milk at room temperature for 1 h. A primary antibody against EGFR (cat. no. ab40815; 1:500; Abcam, Cambridge, UK) and β-tubulin (cat. no. 2128; Cell Signaling Technology Inc., Danvers, MA, USA) was used as the loading control at 4°C overnight. A horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. A0277; 1:2,500; Beyotime Institute of Biotechnology, Shanghai, China) was used as the secondary antibody at room temperature for 2 h. Subsequently, the coloration was completed by DAB (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Images were captured with a Bio-Rad Gel Doc XR and Quantity One v4.6.8 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!