Total protein was extracted using an extraction buffer (Thermo Fisher Scientific, Inc.). The proteins were separated by SDS-PAGE (12% acrylamide gel) and transferred onto a PVDF membrane. The membranes were subsequently blocked with 5% skim milk for 2 h at room temperature to inhibit nonspecific protein binding. This incubation was followed by an incubation with the primary antibodies anti-GAPDH (1:10,000; cat. no. AC002; ABclonal), anti-actin (1:1,000; cat. no. ab8226; Abcam), anti-HMGB1 (1:1,000; cat. no. Ab184532; Abcam), anti-TNF-α (1:1,000; cat. no. ab1793; Abcam), anti-IL-1β (1:1,000; cat. no. A1112; ABclonal), anti-pIKK-β (1:1,000; cat. no. Ab194519; Abcam) and anti-IKK-β (1:1,000; cat. no. A2087; ABclonal) at 4 °C overnight. The secondary antibodies were a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:10,000; cat, no. ab6721; Abcam) and a horseradish peroxidase-conjugated anti-mouse IgG antibody (1:20,000; cat. no. AS003; ABclonal), which were incubated with the membranes for 1 h. The results were imaged and quantitated using Bio-Tanon Imagine (Tanon Science & Technology Co., Ltd., Shanghai, China). ImageJ software (version 1.4; National Institutes of Health, Bethesda, MD, USA) was used to analyse the specific protein band values of each group. All original pictures of western blot results are provided in supplementary file.
Anti ikkβ
Manufactured by Abcam
Sourced in United States
Anti-IKKβ is a primary antibody that binds to and detects the IKKβ (Inhibitor of NF-κB kinase subunit beta) protein. IKKβ is a key component of the IKK complex, which plays a crucial role in the activation of the NF-κB signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti p65, by Abcam (3 mentions)
Anti ikkα, by Abcam (3 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti nf κb p65, by Abcam (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti p p65, by Abcam (2 mentions)
Anti p ikkα β, by Abcam (2 mentions)
Anti hmgb1, by Abcam (1 mentions)
Anti actin, by ABclonal (1 mentions)
Horseradish peroxidase conjugated anti mouse igg antibody, by Abcam (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Abcam (1 mentions)
Extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Anti caspase 1, by GeneTex (1 mentions)
Anti ikk beta, by Abcam (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by BD (1 mentions)
10 protocols using anti ikkβ
1
Protein Extraction and Western Blot Analysis
2
Gingival Fibroblast Inflammatory Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For short-term cultures, gingival fibroblasts were serum-starved for 24 h, then treated with 1 μg/ml LPS for 24 h. For long-term cultures, gingival fibroblasts were treated with 1 μg/ml LPS in α-MEM containing 2% FBS for 14 days. Proteins were detected following a previously published western blot procedure [15 (link)]. The following proteins were detected by the following antibodies: anti-caspase 1 (Cat# 14367; Gene Tex Inc., Irvine, CA), anti-caspase 3 (Cat# 611048; BD Transduction Laboratories, San Jose, CA), anti-IL-1β (Cat# AF-201-NA; R&D Systems Inc., Minneapolis, MN), anti-NLRP3 (Cat#LS-C162911; LifeSpan Biosciences, Inc., Seattle, WA), anti-NF-κB p65 (Cat#3033; phosphorylated Ser 536, Cell Signaling Technology, Inc., MA, USA), anti-NF-κB p65 (Cat# sc-71675; Santa Cruz Biotechnology, Inc., Dallas TX), anti-IKKβ (Cat# ab32041; Abcam, MA), anti-IKK beta (Cat# ab59195; phosphorylated Y199, Abcam), β-actin (Cat# sc-69879; Santa Cruz Biotechnology, Inc.).
3
Assessing Cellular Behavior and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell Counting Kit-8 (CCK-8), Bicinchoninic acid (BCA) Protein Assay Kits, Annexin V-FITC Apoptosis Detection Kits, and Cell Cycle Analysis Kits were all purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Protein Extraction Kits were obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Diaminobenzidine (DAB) substrate kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) kits were provided by Roche Diagnostics (Germany). 4′,6′-Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), and sodium dodecyl sulphate (SDS) were purchased from Sigma (St. Louis, MO, U.S.A.). Anti-LOX1, anti-caspase-9, anti-p65, anti-p-IKKβ, anti-IKKβ, anti-p-IkBα, anti-IkBα, anti-GAPDH, anti-β-tubulin, and anti-Lamin B primary antibodies, and horseradish peroxidase-conjugated antibody were obtained from Abcam (Cambridge, U.K.). Plasmids harboring the wild-type caspase-9 response element (WT-luciferase-caspase-9) and the corresponding mutant (MUT-luciferase-caspase-9) were purchased from Vipotion Biotechnology (Guangzhou, China). Pierce Agarose Chip Kits were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
4
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were loaded onto polyacrylamide gels (8–10%) for electrophoresis. The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. Each membrane was blocked in tris‐buffered saline with Tween 20 (TBST) containing 5% non‐fat dry milk for 1 hr at room temperature and then incubated at 4°C overnight with one of the following primary antibodies: anti‐IKK‐α (1:1000, Abcam, Cambridge, MA, USA); anti‐IKK‐β (1:1000; Abcam); anti‐p‐IKK‐α/β (Ser176/180) (1:1000, CST, Danvers, MA, USA); anti‐NF‐κB‐p65 (1:1000; Abcam); anti‐p‐NF‐κB‐p65 (S536) (1:1000; Abcam); anti‐p38MAPK (1:1000; CST); anti‐p‐p38MAPK (Thr180/Tyr182) (1:1000; CST); anti‐CXCR4 (1:1000; Sigma‐Aldrich); anti‐Bcl‐2 (1:200; CST); or anti‐GAPDH (1:1000; Proteintech). This was followed by incubation in peroxidase‐conjugated AffiniPure goat anti‐rabbit or mouse IgG (H+L) (1:5000, MultiSciences, Hangzhou, Zhejiang, China) for 1 hr at room temperature. Immunoreactivity was detected by chemiluminescence.
5
Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins from blood samples and cells were extracted using RIPA lysis buffer following standard protocols, and protein concentrations were detected using protein assay kit (Bio-Rad Laboratories, Tokyo, Japan). About 30 μg proteins of each sample were separated by SDS-PAGE and then transferred to a PVDF membrane. After blocking for 1 h with 5% skim milk (dissolved in TBST buffer containing 0.1% Tween 20), the membrane was incubated with primary antibodies at 4°C overnight. After that, the membrane was washed with PBS three times and incubated with secondary antibodies at 37°C for 1 h. Blots were detected by enhanced chemiluminescence. The primary antibodies were rabbit antibodies, including anti-NAMPT, anti-p65, anti-p-p65, anti-IKKβ, anti-p-IKKα/β and anti-GAPDH as control (Abcam, Cambridge, U.K.), and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Abcam.
6
Immunoblotting of IKK and NF-κB Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal quantities of cellular proteins, prepared in sample lysis buffer [25 (link)] and heated for 10 min at 100 °C, were electrophoresed through a 10% SDS/polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). The membranes were incubated with anti- PIMREG (also named FAM64A) (1:1000, Cat# ab118102), anti-IKKα (Cat#ab32041), anti- phospho-IKKα (Cat# ab38515), anti-IKKβ (Cat#ab 32135), and anti- phospho-IKKβ (Cat# ab194519) (1:1000, Abcam, Cambridge, MA); and anti-NF-κB/p65 (Cat# 8242), anti- Histone H3 (Cat# 4499), anti-IκBα (Cat# 7543), anti- phospho-IκBα (Cat# 2859), anti-β-actin (Cat# 4970) and anti-α-Tubulin (Cat# 2125), anti- GAPDH (Cat# 5174) antibodies (1:1000; Cell Signaling Technology, Danvers, MA).
7
Immunohistochemical Analysis of Osteosarcoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteosarcoma sections from experimental mice were analyzed via an avidin-biotin-peroxidase technique. Paraffin-embedded tumor tissue sections were prepared and epitope retrieval was performed for further analysis. Paraffin sections were subjected to hydrogen peroxide (3%) for 10–15 min, and subsequently blocked by a regular blocking solution for 10–15 min at 37°C. Finally, the sections were incubated with anti-p65 (cat. no. ab16502; Abcam), anti-IKKβ (cat. no. IMG-129A; Novus Biologicals, LLC, Littleton, CO, USA) and anti-IkBα (cat. no. 9242; Cell Signaling Technology, Inc., Danvers, MA, USA) (all 1:1,000) at 4°C for 12 h. To analyze TRIM-14 expression, tumor sections were stained with DAPI for 60 min at 37°C and incubated with anti-TRIM-14 after washing with PBS three times for 60 min at 37°C. All sections were washed three times with PBS and incubated with peroxidase-labeled antibodies (1:5,000; PV-6013; OriGene Technologies, Inc.) at 37°C for 60 min. From the sections, six random fields of view were observed under a light microscope.
8
Western Blot Analysis of Protein Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After cell lysis, individual proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a gel containing 10% of acrylamide. Trans-blotting onto a nitrocellulose membrane by electrophoresis was performed in 1 × Tris/glycine buffer containing 20% of methanol at 4 °C for 2 h. The membranes were blocked with TBST containing 5% of nonfat dry milk powder for 30 min and then were incubated overnight with the following primary antibodies: anti-IKK-β (Abcam), anti-phospho- (p-)IκBα (Cell Signaling Technology, Danvers, MA, USA), anti-p-p65 (Cell Signaling Technology), anti-p65 (Cell Signaling Technology), and anti-GAPDH (Proteintech, Chicago, IL, USA) at 4 °C. The membranes were washed (three times for 5 min) with TBST and then probed with secondary antibodies for 30 min. After rinsing (three times for 5 min), the bound antibodies were imaged by means of the ECL Detection Kit. The ImageJ software (NIH, Bethesda, MD, USA) was used to quantify intensity of protein bands.
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After removing the supernatant, the cells were washed twice with PBS at 4°C. RIPA lysis buffer (Beyotime, China) containing 1% protease inhibitor (Sigma–Aldrich, USA) and 1% serine protease inhibitor (Sigma–Aldrich, USA) was added for 30 min to lyse cells and extract proteins from each sample. The concentrations of total proteins were detected by a BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (ThermoFisher, USA) at a ratio of 4:1 and boiled at 99°C for 10 min. Samples were loaded and run on SDS–PAGE gels (CWBIO, China) and transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 2% skim milk (BD, USA) for 1 h at room temperature and then incubated with anti-IKKα, anti-IKKβ, anti-pIKKα/β, anti-p65, anti-pp65, anti-p38, anti-pp38, anti-SAPK/JNK, anti-pSAPK/JNK, and anti-GAPDH primary antibodies (Abcam, UK) overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit or goat anti-mouse HRP (Abcam, UK) for 1 hour. Membranes were washed and exposed to chemiluminescent HRP substrate (Millipore, USA). Images were obtained using the GeneGnome XRQ system (Syngene, USA) and analyzed using ImageJ software.
10
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein extraction, cells were lysed in ice-cold immunoprecipitation (IP) buffer containing phosphatase inhibitors, proteinase inhibitors, and phenylmethanesulfonyl fluoride (PMSF). Whole-cell lysates were centrifuged at 10,000 × g for five minutes at 4°C, and the protein concentrations were determined using a BCA protein assay kit. The protein extracts were separated into 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with these primary antibodies: anti-β-actin (60008–1, Proteintech, Wuhan, China), anti-GFP (Protein Tech Group, Inc., Chicago, US), anti-IKKβ (ab178870, Abcam, Cambridge, UK), anti-p-Erk1/2 (#4370, Cell Signaling Technology), anti-Bcl-2 (YM3041, Immunoway), and anti-bax (YT0458, Immunoway). Membranes were incubated for one hour at room temperature with IRDyeTM700DX anti-mouse IgG, IRDyeTM800DX anti-rabbit IgG, or IRDyeTM800DX anti-goat IgG (Rockland, Maine, US) conjugated secondary antibody (1:3000). The bands of interest were detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, US).
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!