Rabbit anti phospho p65

Manufactured by Santa Cruz Biotechnology

Rabbit anti-phospho-p65 is a primary antibody that specifically binds to the phosphorylated form of the p65 subunit of the NF-κB transcription factor complex. This antibody can be used for the detection and quantification of activated NF-κB in various research applications.

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Close spelling variants of this product

Anti-p65 (150 citations) Rabbit anti-p65 (18 citations) Anti-phospho-p65 (10 citations) Phospho-p65 (8 citations) Rabbit anti-TM (2 citations) Rabbit anti-phospho p65 antibody (2 citations)

3 protocols using rabbit anti phospho p65

Ephrin-A1-Fc Signaling Pathway Analysis

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Following treatment with 2 μg/ml ephrin-A1-Fc and/or inhibitors, cells were rinsed with ice cold PBS and processed according to the NE-PER cytoplasmic/nuclear subcellular fractionation kit (Pierce). Nuclear pellets were rinsed with ice cold PBS after extraction of the cytoplasmic fraction and fractions were diluted with 2X Laemmli sample buffer prior to analysis by immunoblotting. Immunoblotting was performed on cell lysates following separation on 10% polyacrylamide gels by SDS-PAGE and subsequent transfer to PVDF membranes. Membranes were blocked with 5% milk in tris-buffered saline containing tween-20, and then probed with rabbit anti-p65, rabbit anti-EphA4, rabbit anti-EphA2, rabbit anti-DSCR1, mouse anti-E-selectin (Santa Cruz); rabbit anti-phospho-p65, rabbit anti-NFAT1, rabbit anti-HDAC3, rabbit anti-β-tubulin, rabbit anti-GAPDH, or rabbit IκBα (Cell Signaling Technologies).

Funk S.D., Finney A.C., Yurdagul A J.r., Pattillo C.B, & Orr A.W. (2018). EphA2 Stimulates VCAM-1 Expression Through Calcium-Dependent NFAT1 Activity. Cellular signalling, 49, 30-38.

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Inflammatory Signaling Pathways in Microglia

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Cultured BV-2 microglia were treated with either 200 or 400 µg/ml sHZ for 24 h. Thereafter, protein was extracted from lyzed cells. Electrophoresis was performed by SDS-PAGE to separate the proteins. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore). Transferred proteins were exposed to rabbit anti-iNOS (1:1000; Cell Signaling), rabbit anti-NLRP3 (1:1000; Abcam), rabbit anti-phospho-p65 (1: 1000; Santa Cruz), and rabbit anti-actin (1:1000; Sigma). Membranes were incubated with the primary antibody overnight at 4 °C. Proteins were detected. Blots were detected with Alexa Fluor^® 680 goat anti-rabbit IgG (Life technologies, UK) using Licor Odyssey imager.

Velagapudi R., Kosoko A.M, & Olajide O.A. (2019). Induction of Neuroinflammation and Neurotoxicity by Synthetic Hemozoin. Cellular and Molecular Neurobiology, 39(8), 1187-1200.

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Western Blot Analysis of Inflammatory Signaling Proteins

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Equal amounts of protein (20 μg) were separated on a polycryamide electrophoresis gel and transferred onto a polyvinylidine fluoride (PVDF) membrane. Membranes were incubated in blocking buffer for 1 h at room temperature, washed 3 times for 10 min each in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and incubated with primary antibodies overnight at 4C. Primary antibodies used were rabbit anti-COX-2 (Santa Cruz;
1:500), rabbit anti-iNOS (Santa Cruz, 1:500), rabbit anti-IBα (Santa Cruz; 1:250), rabbit anti-phospho-IκBα (Santa Cruz, 1:250), rabbit anti-IKKα (Santa Cruz; 1:250), rabbit antiphospho-IKKα (Santa Cruz, 1:250), rabbit anti-phospho-p65 (Santa Cruz, 1:500), rabbit anti-p65 (Santa Cruz 1:500), rabbit anti-microtubule-associated protein-2 (MAP2) (Santa Cruz, 1:500) , rabbit anti-ERβ (Santa Cruz 1:250) and rabbit anti-actin (Sigma Aldrich, 1:500). Primary antibodies were diluted in TBS-T and 1% bovine serum albumin (BSA).
After extensive washing (three times for 15 min each in TBS-T), proteins were detected by incubation with Alexa Fluor 680 goat anti-rabbit secondary antibody (1:10000; Life Technologies) at room temperature for 1 h. Detection was done using a LICOR Odyssey Imager. All Western blot experiments were carried out at least three times.

El-Bakoush A, & Olajide O.A. (2018). Formononetin inhibits neuroinflammation and increases estrogen receptor beta (ERβ) protein expression in BV2 microglia. International immunopharmacology, 61.

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