Total protein was extracted from RA-FLS using the protein extraction kit (Vazyme Biotech Co., Ltd.) and protein concentration was quantified using the BCA assay method. Proteins were separated via 10% SDS-PAGE, transferred onto PVDF membranes (MilliporeSigma) and blocked with 5% non-fat milk in 0.05% TBS-Tween-20 for 1 h at room temperature. The membranes were incubated with primary antibodies against IκB (1:1,000; cat. no. 4812; Cell Signaling Technology, Inc.), phosphorylated (p)-IκB (1:1,000; cat. no. 2859; Cell Signaling Technology, Inc.), NF-κB (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-NF-κB (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. 2118; Cell Signaling Technology, Inc.) overnight at 4˚C. Following the primary antibody incubation, the membranes were incubated with the HRP-conjugated goat anti-rabbit polyclonal IgG secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.). Protein bands were visualized by Pierce ECL Plus Western Blotting substrate (Thermo Fisher Scientific, Inc.) and subsequently analyzed using ImageJ software (version 1.51j8; National Institutes of Health). GAPDH was used as the internal control.
Phosphorylated iκb
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphorylated (p)-IκB is a protein that has been modified by the addition of a phosphate group. IκB is an inhibitor of the NF-κB transcription factor, and its phosphorylation leads to the activation of NF-κB and the subsequent regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Nf κb, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protein extraction kit, by Vazyme (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Iba 1, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection system, by Merck Group (1 mentions)
Sirt1, by Abcam (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (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)
Total iκb, by Cell Signaling Technology (1 mentions)
4 protocols using phosphorylated iκb
1
Western Blot Analysis of NF-κB Signaling
2
Western Blot Analysis of Apoptosis and Inflammation Markers
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Proteins were extracted with RIPA lysis buffer (Santa Cruz Biotechnology), and 30 μg of total protein was loaded on a gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and probed with primary antibodies against cleaved caspase-3 (1:300, Cell Signaling Technology), Bax (1:500, Abcam), SIRT1, Iba-1, phosphorylated (p)-IκB, TLR4, and NF-κB p65 (1:200, Cell Signaling Technology), followed by incubation with appropriate horseradish peroxidase-conjugated IgG (1:5000, Boster Biotech) secondary antibodies. Immunoblots were visualized using the Millipore ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or Lamin B1 (1:3000, Cell Signaling Technology).
3
Epac and NF-κB Regulation in Retina
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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.
4
Nrf2 Signaling Pathway Analysis
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Antibodies for Nrf2, phosphorylated IKK, IKK-α, IKK-β, phosphorylated IκB, p65, phosphorylated JNK, total JNK, phosphorylated p38, total p38, phosphorylated ERK1/2, total ERK1/2, HO-1, an FITC-conjugated secondary antibody, and U0126 (ERK1/2 inhibitor) were purchased from Cell Signaling Technology (Beverley, MA, USA). A GCLM antibody and SB202190 (p38 inhibitor) were ordered from Abcam (Cambridge, UK), an iNOS antibody was supplied by BD Biosciences (Franklin Lakes, CA, USA), an actin antibody was purchased from Millipore (Bedford, MA, USA), and horseradish peroxidase–conjugated secondary antibodies were provided by Santa Cruz Biotechnologies (Santa Cruz, CA, USA). A protease inhibitor cocktail, a phosphatase inhibitor cocktail, and SP600125 (a JNK inhibitor) were supplied by Calbiochem (Merck Millipore, Darmstadt, Germany). Cell Culture Lysis Reagent was purchased from Promega (Madison, WI, USA). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). Fetal bovine serum was acquired from Invitrogen (Carlsbad, CA, USA). Cell culture media, dimethylsulfoxide (DMSO), and other chemicals were ordered from Sigma Chemical Company (St. Louis, MO, USA).
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