Whole cell lysates were prepared by boiling cell pellets for 10 minutes in SDS lysis buffer [2% SDS, 10% Glycerol, 62 mmol/L Tris-HCl, pH 6.8 and a complete mini-protease inhibitor cocktail (Roche Applied Science)]. After protein quantification with Bio-Rad Dc Protein Assay (Bio-Rad Laboratories), equal amounts of proteins were loaded, separated on a polyacrylamide gel, and transferred to a nitro-cellulose membrane. Protein bands were immunodetected with appropriate antibodies, e.g., goat anti-DDB2 (R&D Systems), rabbit anti-Nanog (Cell Signaling Technology), mouse anti-Tubulin (Millipore), and mouse anti-IκBα (Cell Signaling Technology).
Mouse anti iκbα
Manufactured by Cell Signaling Technology
Sourced in United States
Mouse anti-IκBα is a primary antibody that specifically binds to the IκBα protein. IκBα is an inhibitor of the NF-κB transcription factor, playing a role in the regulation of inflammatory and immune responses. This antibody can be used to detect and study the expression and localization of IκBα in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Merck Group (2 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (2 mentions)
Rabbit anti ampk, by Cell Signaling Technology (2 mentions)
Rabbit anti p65, by Cell Signaling Technology (2 mentions)
Rabbit anti erk1 2, by Cell Signaling Technology (2 mentions)
Rabbit anti p iκbα, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Mouse anti tubulin, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Rabbit anti nanog, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini instrument, by GE Healthcare (1 mentions)
Rabbit igg antibodies, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Mouse anti e cadherin, by Cell Signaling Technology (1 mentions)
Goat anti gli3, by R&D Systems (1 mentions)
14 protocols using mouse anti iκbα
1
Whole Cell Lysis and Western Blot
2
Immunoblotting Protocol for Analyzing Cell Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells for immunoblotting were prepared as we described previously (22 (link)). Briefly, Cells were solubilized in lysis buffer (1.0% NP-40, 50 mM HEPES, pH7.4, 150 mM NaCl), containing protease and phosphatase inhibitor (Thermo fisher scientific). Cell lysates were separated by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), transferred to a Polyvinylidene difluoride (PVDF) membrane (Merck), and detected with the following antibodies using ECL substrate (Bio-Rad): rabbit anti-CCAAT enhancer binding protein (C/EBP)α anti-phospho-C/EBPα (Ser21), anti-phospho- C/EBPα (Thr221/226), anti-Erk1/2, anti-phospho- Erk1/2, anti-JNK, anti-phospho-JNK, anti-NF-κB(p65), anti-phospho-NF-κB(p65), mouse anti-IκBα (Cell Signaling Technology), mouse anti-IκBβ (Santa Cruz Biotechnology), mouse anti-βactin (Sigma-aldrich), horseradish peroxidase (HRP)-conjugated mouse IgG (Cell Signaling Technology), or rabbit IgG antibodies (Cell Signaling Technology). Digital images were obtained using an ImageQuant LAS4000 mini instrument (GE Healthcare). Densitometry was performed on scanned blots using the ImageQuant TL software program (GE Healthcare).
3
Protein Expression Analysis in Transfected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells or grinded tumor tissues were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche complete Protease Inhibitor Cocktail) for 1 h at 4 ℃. The lysate was clarified by centrifugation for 20 min at 14,000 × g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 5 × SDS loading buffer, incubated at 95 ℃ for 5 min, subjected to SDS-PAGE, followed by western blot analysis. The following antibodies were used: rabbit anti-β-actin (Affinity; 1:5000), rabbit anti- AMPKα (Cell Signaling Technology; 1:1000), rabbit anti-Phospho-AMPKα (Cell Signaling Technology; 1:1000), mouse anti-E-cadherin (Cell Signaling Technology; 1:1000), rabbit anti-N-cadherin (Elabscience; 1:1000), rabbit anti-MMP3 (Proteintech; 1:1000), rabbit anti-GLI1 (Cell Signaling Technology; 1:1000), rabbit anti-GLI2 (NOVAS; 1:1000), goat anti-GLI3 (R&D; 1:1000), mouse anti-IκBα (Cell Signaling Technology; 1:1000), rabbit anti-NF-κB p65 (Cell Signaling Technology; 1:1000) and rabbit anti-Phospho-NF-κB p65 (Cell Signaling Technology; 1:1000).
4
Western Blot Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues and cell samples were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL) containing a protease inhibitor cocktail (Calbiochem, Raleigh, NC). Protein concentrations were quantified with a BCA protein assay kit (Thermo Scientific, Rockford, IL) according to the manufacturer's manual. Fifty micrograms of total protein from each tissue or cell lysate sample were loaded and separated by gel electrophoresis and then transferred to nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies at 4 °C overnight under shaking conditions. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Detection and quantification of protein bands were performed using the ChemiDocTM XRS+ System with Image LabTM Software (Bio-Rad). The primary antibodies used were rabbit anti-phospho-Stat3 (Tyr705), rabbit anti-Stat3, rabbit anti-phospho-IκBα (Ser32), mouse anti-IκBα (Cell Signaling), anti-β-actin (Sigma).
5
Investigating Inflammatory Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aze was purchased from Selleckchem (Houston, TX, USA). SP600125 was obtained from Calbiochem (Darmstadt, Germany). LPS (Escherichia coli, serotype 011:B4), mouse anti-β-actin (1:2000, cat# A5316) and rabbit anti-lamin B1 (1:2000, cat# SAB1306342) antibodies, dimethyl sulfoxide (DMSO), and adenylyl-imidodiphosphate (AMPPNP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (IgG) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Thermo Scientific (Rockford, IL, USA). Antibiotic-antimycotic reagent, Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). The rabbit anti-phospho-IκBα (1:1000, cat# ab97783) antibody was supplied by Abcam (Cambridge, MA, USA). Rabbit anti-phospho-JNK (1:1000, cat# 4668), rabbit anti-JNK3 (1:1000, cat# 2305), rabbit anti-phospho-c-Jun (1:1000, cat# 9261), rabbit anti-c-Jun (1:1000, cat# 9165), rabbit anti-iNOS (1:1000, cat# 2982), rabbit anti-COX2 (1:1000, cat# 4842), mouse anti-NF-κB p65 (1:1000, cat# 6956), mouse anti-IκBα (1:1000, cat# 4814) antibodies, and horseradish peroxidase-conjugated anti-rabbit (1:2000, cat# 7074) and anti-mouse IgG (1:2000, cat# 7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Adiponectin Receptor and AMPK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell homogenates (20 μg/well) were loaded onto 10% SDS polyacrylamide gels in denaturing conditions at 80 mA for 90 min and transferred electrophoretically (100 mA/blot, 2 h; Power Pack; Bio-Rad Laboratories, Inc., USA) to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was performed as described previously [48 (link)]. Nonspecific binding was blocked with 5% non-fat milk powder in Tris-buffered saline-Tween containing 0.1% Tween-20 (PBS-T) for 1 h. Primary antibodies including rabbit anti-AdipoR1 (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-AdipoR2 (1:1000, Boster Biological Technology, USA), rabbit anti-AMPK (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-AMPKT172 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-α-Tubluin (1:5000, Cell Signaling Tech. Inc., USA), rabbit anti-p-NF-κB p65S536 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-NF-κB p65 (1:1000, Cell Signaling Tech. Inc., USA), rabbit anti-p-IκBα (Ser32) (1:1000, Cell Signaling Tech. Inc., USA), mouse anti-IκBα (1:1000, Cell Signaling Tech. Inc., USA) antibody were incubated at 4 °C overnight, followed by HRP-conjugated secondary antibodies (goat anti-rabbit, 1:5000 or rabbit anti-mouse, 1:5000; Dako, Glostrup, Denmark) at RT for 1 h. The immunoblot signals were visualized by Westernbright Quantum HRP substrate (advansta, USA).
7
Western Blot Analysis of Cardiac Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as we described previously [18] (link). The border zone of the infarct hearts was separated and homogenized. Equal amounts of protein (30 mg) were separated on SDS–polyacrylamide gels (10%) and electrotransferred to polyvinylidene difluoride membranes (Roche, Switzerland). The membranes were incubated with rabbit anti-LC3 (1∶1000 dilution; Cat#L7543, Sigma), rabbit anti-Beclin-1 (1∶1000 dilution; Cat#3738, Cell signaling technology), rabbit anti-p65 (1∶1000 dilution; Cat#9936S, Cell signaling technology), mouse anti-IκBα (1∶1000 dilution; Cat#9936S, Cell signaling technology), rabbit anti-P62(Cat#5114 Cell signaling technology), rabbit anti-AMPK (Cat#5831, Cell signaling technology), rabbit anti-Phospho-AMPK (Cat#2535, Cell signaling technology), mouse anti-HIF-1α (NB100-105, Novus Biologicals), goat anti-GAPDH and goat anti-β-actin (1∶1000 dilution; Cat#SC-48166 and Cat#SC-1616, Santa Cruz Biotechnology) at 4°C overnight, and incubated with either goat anti-rabbit, rabbit anti-mouse or mouse anti-goat second antibody (1∶5000 dilution; Santa Cruz Biotechnology) for 1 hour at room temperature. Blots were developed using a chemiluminescent substrate and molecular band intensity was determined by densitometry.
8
Protein Expression Analysis in Disc Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nucleus pulposus cells and disc tissues lysates were prepared using RIPA buffer (Beyotime Biotechnology, Shanghai, China). Nuclear proteins were extracted using a nuclear protein extraction kit (BestBio, Shanghai, China). After detected the total protein concentrations by a bicinchoninic acid (BCA) protein assay (Beyotime, China), an equal amount of protein was resolved on 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblot analyses. The following primary antibodies were used to incubate proteins overnight at 4°C: goat anti-FSTL1, rabbit anti- iNOS, rabbit anti-TNF-α, rabbit anti-MMP-13, rabbit anti- COX-2 (all 1:2000; Abcam, USA); rabbit anti-p38, rabbit anti-p-p38, rabbit anti- JNK, rabbit anti-p-JNK, rabbit anti- ERK1/2, rabbit anti-p-ERK1/2, rabbit anti-p65, mouse anti- IκBα, rabbit anti-p-IκBα (all 1:1000; Cell Signaling Technology, Inc., MA, USA). The protein bands on the PVDF membranes were washed then horseradish peroxidase-conjugated secondary antibody (1:10000) was added and the bands were visualized using a FluorChem E chemiluminescent imaging system (Amersham Imager 600, General Electric Company, USA). ImageJ software (National Institutes of Health, USA) was used for densitometry analysis.
9
Prostate Tissue Protein Expression Analysis
10
Western Blotting Analysis of Cellular and Tissue Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting analysis was performed as previously described.25 (link) Cellular and mouse tissue proteins were exposed to radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.6], 150 mmol/L NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS), homogenized on ice and centrifuged. The supernatants were resolved via 10% SDS‐PAGE and were transferred to PVDF membranes (Millipore). The membranes were blocked with Tris‐buffered saline (TBS) containing 5% non‐fat milk and were incubated with the following primary antibodies: rabbit anti‐IRF7 (sc‐9083; 1:200; Santa Cruz), mouse anti‐PCNA (#2586; 1:1000; Cell Signaling Technology), rabbit anti‐Cyclin D1 (#2978; 1:1000; Cell Signaling Technology), rabbit anti‐NF‐κB p65 (#4764; 1:1000; Cell Signaling Technology), rabbit anti‐phosphorylated‐NF‐κB p65 (ser536) (BS4138; 1:1000; Bioworld), mouse anti‐IκBα (#4814; 1:1000; Cell Signaling Technology), mouse anti‐phosphorylated IκBα (BS4105; 1:1000; Bioword), rabbit anti‐TNFα (#3707; 1:1000; Cell Signaling Technology), goat anti‐IL‐6 (AF‐406‐NA; 1:500; R&D System), goat anti‐Collagen type I (sc‐8784; 1:1000; Santa Cruz), goat anti‐Collagen type III (sc‐8781; 1:1000; Santa Cruz), rabbit anti‐ATF3 (sc‐188; 1:200; Santa Cruz), and mouse anti‐GAPDH (MB001; 1:10 000; Bioworld).
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!