The compounds used in this study were dissolved directly in DMEM and the pH corrected to pH7.4. The phospho-acetyl-CoA carboxylase (ACC) Ser 79 antibody was from the Division of Signal Transduction Therapy at the University of Dundee. The total ACC, total AMPKα, phospho-AMPKα Thr 172, total S6, phospho-S6 Ser 240/244, phospho-p70S6K Thr 389, total IκB, pNF-κB, total IKKα, and total IKKβ antibodies for immunoblotting were from Cell Signaling Technology. Actin antibody was from Merck. Antibodies used in the AMPK activity assays were a generous gift from Prof D. Grahame Hardie at the University of Dundee. Chemical structures were drawn using ChemSketch. BI605906 was a generous gift from Prof Sir Philip Cohen (MRC Protein Phosphorylation and Ubiquitylation Unit, Dundee).
Total ikkβ
Manufactured by Cell Signaling Technology
Sourced in United States
Total IKKβ is a lab equipment product that measures the total amount of the IKKβ (Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Beta) protein in a sample. IKKβ is a key component of the NF-κB signaling pathway, which regulates various cellular processes. This product provides a quantitative assessment of the IKKβ levels, without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Total iκbα, by Cell Signaling Technology (3 mentions)
Phospho iκbα, by Cell Signaling Technology (2 mentions)
Phospho nf κb p65, by Cell Signaling Technology (2 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Total ampkα, by Cell Signaling Technology (1 mentions)
Total s6, by Cell Signaling Technology (1 mentions)
Phospho s6 ser240 244, by Cell Signaling Technology (1 mentions)
Total acc, by Cell Signaling Technology (1 mentions)
Total iκb, by Cell Signaling Technology (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Total ikkα, by Cell Signaling Technology (1 mentions)
Phospho p70s6k thr389, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Total nf κb p65, by Cell Signaling Technology (1 mentions)
β mhc sc53090, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Glut2, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using total ikkβ
1
Phospho-ACC and AMPK Pathway Analysis
2
Antibody Detection of Cardiac Signaling Proteins
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Antibodies against the following proteins were purchased from Santa Cruz Biotechnology (Dallas, TX): atrial natriuretic peptide (ANP, sc20158 1:200), β‐myosin heavy chain (β‐MHC, sc53090 1:200), and SNIP1 (sc47929 1:200). Antibody against phospho‐IKKβ (ab59195 1:500) was purchased from Abcam Biochemicals (Cambridge, MA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): phospho‐NF‐κB p65 (#3033 1:1000), total‐NF‐κB p65 (#4764 1:1000), total‐IKKβ (#8943 1:1000), phospho‐IκBα (#2859 1:1000), total‐IκBα (#4812 1:1000), cleaved caspase‐3 (#9661 1:1000), and GAPDH (#2118 1:1000). Fetal bovine serum was purchased from Gibco (Grand Island, NY). The other reagents for cell culture were purchased from Sigma (St. Louis, MO).
3
Immunoblotting Analysis of Signaling Pathways
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Immunoblotting was performed as described previously (Xie et al., 2015 (link)). The primary antibodies included total‐IKKβ, phosphorylation‐IKKα/β, total and phosphorylation‐IκBα, total and phosphorylation P65 (Cell Signaling Technology, MA, USA), SCD1, GLUT2, PPARγ, and β‐actin (Santa Cruz, CA, USA). Image J software was used to analyze the intensity of protein expression.
4
Western Blotting Protocol for Protein Analysis
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Western-blotting was performed with precast gradient gels (Bio-Rad) using standard methods as described previously [36 (link), 37 (link)]. Briefly, total protein from each sample was resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylimide gel electrophoresis and was transferred to the Immobilon™ PVDF Transfer Membranes (Millipore Corporation, Billerica, MA). The membrane was then blocked in 5% bovine serum albumin (BSA) and incubated with the primary antibodies against CYLD (1:1000, Cell Signaling Technology, USA), phospho-IKKβ (1:1000, Cell Signaling Technology), total IKKβ (1:1000, Cell Signaling Technology), total IκBα (1:1000, Cell Signaling Technology), RelA (1:1000, Cell Signaling Technology), Flag (1:1000; ProteinTech group, USA), proliferating cell nuclear antigen (PCNA) (1:2000, Biosynthesis, China) and GAPDH (1:3000, Biosynthesis). After incubation with HRP-linked secondary antibodies, the bands were visualized by western chemiluminscent HRP Substrate Kit (PPLYGEN, Beijing, China).
5
IGPR-1 Phosphorylation Regulates Autophagy
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Anti–IGPR-1 and anti-pSer220 antibodies are homemade rabbit polyclonal antibodies previously described (15 (link), 16 (link)). Phospho-AMPK (Thr172), total AMPK, phospho-ULK1 (Ser555), total ULK1, phosph-Beclin-1 (Ser93), total Beclin-1, phospho-IKK (Ser176/180), total IKKβ, and LC3A/B, GAPDH antibodies all were purchased from Cell Signaling Technologies (Danvers, MA, USA). The following plasmids were all purchased from Addgene (Watertown, MA, USA): pcDNA3.FLAG-ULK1 (catalog no. 27636), pMRX-IP-GFP-LC3-RFP (catalog no. 84573), pcDNA-IKKβ-FLAG (catalog no. 23298), pcDNA-IKKβ-A44 (catalog no. 23299), and constitutive active IKKβ (S177E S181E, catalog no. 11105). IGPR-1 constructs including WT IGPR-1 and Ser220 mutant, A220–IGPR-1 constructs were cloned into retroviral vector, pQCXIP with C-terminal Myc tag as previously described (16 (link), 17 (link), 18 (link)). Retroviruses were produced in 293-GPG cells as described (41 (link)). IKK inhibitor III and rapamycin were purchased from Calbiochem, and LPS was purchased from Sigma. Oligomycin was purchased from Cell Signaling Technologies. A set of three human IKKβ sgRNAs (catalog no. GSGH-11938-16EG3551) were purchased from Dharmacon (Chicago, IL, USA).
6
Signaling Pathway Analysis by Western Blot
7
Oxidative Stress Signaling Pathway
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Superoxide dismutase (SOD) and catalase were purchased from Sigma-Aldrich (St. Louis, MO). Protease inhibitor and phosphatase inhibitor cocktail tablets were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Antibodies for phospho-p38 (#4631), total p38 (#9228), phospho-SAPK/JNK (#9255), total SAPK/JNK (#9252S), phospho-p44/42 (#8544), total p44/42 (#4695), phospho-IKKα/β (#2697), total IKKβ (#8943), total IκB (#4814), phospho-cJun (#3270), total cJun (#9165), phospho-NFκB p65 (#3033), total NFκB p65 (#8242) were purchased from Cell Signaling Technology (Danvers, MA). VCAM-1 (sc-13160), ICAM-1 (sc-19584), and β-Actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Nox2 antibody (ab129068) was purchased from Abcam (Cambridge, MA) and p47phox antibody (07-001) was purchased from Millipore (Burlington, MA). Rabbit (925-68070), mouse (925-68071) and goat (925-68074) secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). The nuclear extract kit (40010) was purchased from Active Motif (Carlsbad, CA). Coumarin 7-boronic acid (CBA) (1357078-03-5) was purchased from Cayman Chemical (Ann Arbor, MI). Hydropropidine (HPr+) was generously provided by Dr. Jacek Zielonka (Medical College of Wisconsin).
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