After 24 h of co-culture, RAW264.7 cells were collected from the Transwell chamber and total proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.; cat. no. 89900). Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane, which were then incubated with primary antibodies for NF-kB p65 (1:700; Affinity Biosciences; cat. no. AF5006), Phospho-NF-kB p65 Ser536 (1:500; Affinity Biosciences; cat. no. AF2006), TYROBP (1:1,000; Abcam; cat. no. ab280568) and GAPDH (1:500; Affinity Biosciences; cat. no. AF7021) overnight at 4°C. The membrane was then treated with a horseradish peroxidase-conjugated secondary antibody (1:1,000; Affinity Biosciences; cat. no. S0001) for 2 h at room temperature. After washing with PBS, the membrane was developed in an X-ray film cassette, and the films were scanned and analyzed using QuantityOne software (version 4.6.8; Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control for normalizing target protein expression. Each experiment was performed in triplicate.
Phospho nf kb p65 ser536
Manufactured by Affinity Biosciences
The Phospho-NF-kB p65 Ser536 is a laboratory reagent used to detect and quantify the phosphorylation of the NF-kB p65 subunit at the serine 536 residue. This assay provides a tool to monitor the activation of the NF-kB signaling pathway, which is involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Affinity Biosciences (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Ab109461, by Abcam (1 mentions)
Af2006, by Affinity Biosciences (1 mentions)
Af5002, by Affinity Biosciences (1 mentions)
A19684, by ABclonal (1 mentions)
Af2002, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
2 protocols using phospho nf kb p65 ser536
1
Western Blot Analysis of NF-kB Signaling
2
Protein Expression Analysis of Neuroinflammation
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The BV2 or SH-SY5Y cells or midbrain tissue lysates were subjected to Western blotting to detect NFATc2, STAT1, p-STAT1, IRF9, BAX, BCL-2, p65, p-p65, IκBα, p-IκBα and GAPDH. The following antibodies were used for protein detection by western blotting. NFATc2 (22023–1-AP, 1:1000, Proteintech), STAT1 (66545–1-lg, 1:2000, Proteintech), p-STAT1 (ab109461, 1:1000, Abcam), IRF9 (14167–1-AP, 1:1000, proteintech), BAX (A19684, 1:1000, ABclonal), BCL-2 (26593–1-AP, 1:1000, Proteintech), NF-kB p65 (AF5006, 1:1000, Affinity), Phospho-NF-kB p65(Ser536) (AF2006, 1:1000, Affinity), IKB alpha (AF5002, 1:1000, Affinity), Phospho-IKB alpha (Ser32/Ser36) (AF2002, 1:1000, Affinity), and GAPDH (#5174, 1:1000, Cell Signaling Technology).
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