Phospho rela ser536

Manufactured by Cell Signaling Technology

Phospho-RelA (Ser536) is a lab equipment product that detects the phosphorylation of the RelA subunit of the NF-kB transcription factor at serine 536. This phosphorylation event is associated with the activation of the NF-kB signaling pathway.

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Lab products found in correlation

Odyssey imaging system, by LI COR (1 mentions) Irdye 800 coupled secondary antibodies, by LI COR (1 mentions) Nucleolin sc 13057, by Santa Cruz Biotechnology (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions) Ecl plus, by GE Healthcare (1 mentions) β actin 8h10d10, by Cell Signaling Technology (1 mentions) Rela p65, by Cell Signaling Technology (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Phospho stat1 tyr701, by Cell Signaling Technology (1 mentions) Mhc 1, by Thermo Fisher Scientific (1 mentions) Nf κb2 p100 52, by Cell Signaling Technology (1 mentions) Nf κb1 p105 50, by Cell Signaling Technology (1 mentions) Stat1, by Cell Signaling Technology (1 mentions) Pd l1, by Abcam (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

Phospho-p65/RelA (Ser536) (5 citations)

3 protocols using phospho rela ser536

Dissection and Analysis of Nuclear Proteins

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Cell extracts were prepared using buffer (150 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM EDTA,10 mM NaF, 1 mM Na₃VO₄, 0.5% NP-40, 2% sodium dodecyl sulfate, 10% glycerol, 10 mM DTT, 1 mM PMSF, protease inhibitor cocktail (Roche)). Nuclei were isolated using nuclear lysis buffer A (20 mM Tris-HCl pH 8.0, 10 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, protease inhibitor cocktail) followed by centrifugation at 16000 g for 20 s. Nuclear pellets were sonicated in lysis buffer B (20 mM Tris-HCl pH 8.0, 400 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, protease inhibitor cocktail). Protein lysates were separated on polyacrylamide gels followed by transfer onto nitrocellulose membranes. Membranes were incubated with antibodies against phospho-IKK (Ser176/Ser180) (#2697), phospho-p38 MAP kinase (Thr180/Tyr182) (#9211), phospho-ERK (Thr202/Tyr204) (#9101), phospho-cJun (Ser73) (#3270), IKKβ (#8943), ERK (#4696), p38 MAP kinase (#9212), RelA (#8242), phospho-RelA (Ser536) (#3033), IκB (#4814) (all Cell Signalling Technology) or nucleolin (sc-13057) (Santa Cruz Biotechnology) followed by IRDye 800-coupled secondary antibodies (LICOR Biosciences). Blots were visualized and quantified using the Odyssey imaging system (LICOR Biosciences).

Kirchner J., Brüne B, & Namgaladze D. (2018). AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages. Scientific Reports, 8, 7801.

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Quantifying Phosphorylated Signaling Proteins

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Proteins were extracted by lysed tumor cells using RIPA lysis and extraction buffer (Thermo Fisher Scientific). The western blot analysis was used to determine the expression of total and phosphorated proteins. The intensity of protein bands was detected using ECL Plus (GE Healthcare Life Sciences) using the ChemiDoc Imaging System (Bio-Rad). The antibodies targeting Phospho-IκBα (Ser32) (clone 14D4, #9246), IκBα (clone 44D4, #4812), Phospho-RELA (Ser536, #3033), RELA (clone D14E12, #8242) and β-actin (8H10D10, #3700) were purchased from the Cell Signaling Technology and used for western blot analysis.

Hou J., Wang Y., Shi L., Chen Y., Xu C., Saeedi A., Pan K., Bohat R., Egan N.A., McKenzie J.A., Mbofung R.M., Williams L.J., Yang Z., Sun M., Liang X., Rodon Ahnert J., Varadarajan N., Yee C., Chen Y., Hwu P, & Peng W. (2021). Integrating genome-wide CRISPR immune screen with multi-omic clinical data reveals distinct classes of tumor intrinsic immune regulators. Journal for Immunotherapy of Cancer, 9(2), e001819.

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Western Blot Analysis of Immune Regulators

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Cells were lysed in RIPA buffer (Thermo Fisher, 89900) supplemented with phosphatase and protease inhibitors and subjected to SDS-PAGE. Antibodies used were ERa (Santa Cruz Biotechnology; cat. #sc-543, RRID:AB_631471), RelA/p65 (Cell Signaling Technology; cat. #6956, RRID:AB_10828935), phospho-RelA Ser536 (Cell Signaling Technology; cat. #3033, RRID:AB_331284), GAPDH (Santa Cruz Biotechnology; cat. #sc-25778, RRID:AB_10167668), RelB (Cell Signaling Technology; cat. #10544, RRID:AB_2797727), NF-κB1 p105/50 (Cell Signaling Technology; cat. #12540, RRID:AB_2687614), NF-κB2 p100/52 (Cell Signaling Technology cat. #3017, RRID:AB_10697356), IRF1 (Cell Signaling Technology; cat. #8478, RRID:AB_10949108), STAT1 (Cell Signaling Technology; cat. #9176, RRID:AB_2240087), phospho-STAT1 Tyr701 (Cell Signaling Technology; cat. #9167, RRID:AB_561284), MHC-I (Thermo Fisher Scientific; cat. #MA5-11723, RRID:AB_10985125), PD-L1 (Abcam; cat. #ab228415, RRID:AB_2884993).

Hermida-Prado F., Xie Y., Sherman S., Nagy Z., Russo D., Akhshi T., Chu Z., Feit A., Campisi M., Chen M., Nardone A., Guarducci C., Lim K., Font-Tello A., Lee I., García-Pedrero J., Cañadas I., Agudo J., Huang Y., Sella T., Jin Q., Tayob N., Mittendorf E.A., Tolaney S.M., Qiu X., Long H., Symmans W.F., Lin J.R., Santagata S., Bedrosian I., Yardley D.A., Mayer I.A., Richardson E.T., Oliveira G., Wu C.J., Schuster E.F., Dowsett M., Welm A.L., Barbie D., Metzger O, & Jeselsohn R. (2023). Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor–Positive Breast Cancer. Cancer Research, 83(19), 3284-3304.

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