Whole retinal lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated on a precast Tris-glycine gel (Invitrogen) and blotted on nitrocellulose membrane. After blocking in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and 5% (w/v) bovine serum albumin, the membranes were treated with Epac1 (ab109415), Epac 2 (ab193665, Abcam), total nuclear factor kappa beta (NFκB; #4764), phosphorylated NFκB (Ser 536, #3303), phosphorylated IκB (Ser32, #2859), total IκB (#4812, Cell Signaling, Danvers, MA), and beta actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected with a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA), and data were acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.
Total iκb
Manufactured by Cell Signaling Technology
Sourced in United States
Total IκB is a laboratory equipment product that measures the total amount of the IκB protein in a sample. IκB is a key regulator of the NF-κB signaling pathway, which plays a role in various cellular processes. The Total IκB product provides a quantitative assessment of the IκB protein levels, without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Phosphorylated iκb, by Cell Signaling Technology (1 mentions)
Chemiluminescence reagent kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ab109415, by Abcam (1 mentions)
Ab193665, by Abcam (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Total acc, 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 ikkα, by Cell Signaling Technology (1 mentions)
Total ikkβ, by Cell Signaling Technology (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Phospho p70s6k thr389, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
4 protocols using total iκb
1
Epac and NF-κB Regulation in Retina
2
Phospho-ACC and AMPK Pathway Analysis
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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).
3
Western Blotting of Cellular Proteins
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Total cellular proteins were extracted and resolved on 10% SDS–polyacrylamide gels before transferring to Immobilon-FL PVDF membranes (Millipore, Bedford, MA, USA) as previously described [15 (link)]. Western blots were probed with the following primary antibodies targeting: MITF (1:1000, C5, Cat no: OP126L; Calbiochem, San Diego, CA, USA), AXL (1:200, Cat no: AF154; R&D Systems, Minneapolis, MN, USA,), Melan A (MART-1, 1:1000, Cat no: 34511; Cell Signaling, Danvers, MA, USA), total IκB (1:1000, Cat no: 4812; Cell Signaling), and ß-actin (1:6000, Cat no: A5316; Sigma-Aldrich) overnight at 4 °C. Membranes were detected on the ChemiDoc MP imaging system (BioRad, Hercules, CA, USA) and densitometry quantification performed using the Image Lab software (BioRad).
Densitometric values for each protein were normalized to ß-actin and values were converted to z-scores to enable the analysis of independent Western blotting experiments.
Densitometric values for each protein were normalized to ß-actin and values were converted to z-scores to enable the analysis of independent Western blotting experiments.
4
Quantification of Renal Injury Markers
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Total proteins were prepared from cortical kidney tissue or cultured cells using RIPA lysis buffer (Millipore, Bedford, MA, U.S.A.). Equal amount of proteins were resolved in 4–12% SDS-PAGE gel (Invitrogen), transferred to a PVDF membrane (Millipore). After blocking, membrane was incubated overnight with primary antibodies against fibronectin (Sigma), KIM-1 (R&D Systems, Minneapolis, MN, U.S.A.), TLR4 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), p-IκB, total IκB, p-p38, and p38 (Cell Signaling Technology, Beverly, MA, U.S.A.). The immunocomplex was visualized with ECL prime chemiluminescence (Bio-Rad, Hercules, CA, U.S.A.) using the ChemiDoc XRS system (Bio-Rad). Quantification of protein bands was performed by the ImageJ program (NIH) and was normalized to GAPDH level.
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