P65 8242

Manufactured by Cell Signaling Technology

Sourced in United States

The P65 (8242) is a primary antibody product offered by Cell Signaling Technology. It is designed to detect the p65 subunit of the NF-κB transcription factor complex.

Automatically generated - may contain errors

Lab products found in correlation

Iκbα 4814, by Cell Signaling Technology (2 mentions) Actin 1 19, by Santa Cruz Biotechnology (1 mentions) Actin 3700, by Cell Signaling Technology (1 mentions) H3k27me3, by Merck Group (1 mentions) Immobilon p membrane, by Merck Group (1 mentions) Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Timp 3 ab39184, by Abcam (1 mentions) Anti β actin a5441, by Merck Group (1 mentions) Anti hmgb1 ab79823, by Abcam (1 mentions) P p65 s536 3033, by Cell Signaling Technology (1 mentions) Csb el015893hu, by Cusabio (1 mentions) His tag, by Cell Signaling Technology (1 mentions) Csb e16539 m, by Cusabio (1 mentions) Elisa kit, by Cusabio (1 mentions) Anti mouse igg hrp ap308p, by Merck Group (1 mentions) Anti c myc ht101, by Transgene (1 mentions) Anti lmnb1 101237 t32, by Sino Biological (1 mentions) Anti stub1 12496 r034, by Sino Biological (1 mentions) Hrp anti hemagglutinin 12013819001, by Roche (1 mentions)

6 protocols using p65 8242

Molecular Mechanisms of Withaferin A-Induced Cell Signaling

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Withaferin A and gram-negative bacterial lipopolysaccharide (LPS) were purchased from Calbiochem (Darmstadt, Germany). Recombinant human and mouse TNFα and recombinant human IL-1β was purchased from EMD Millipore (Darmstadt, Germany). Etoposide (VP-16) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against NEMO (FL-419), Ubiquitin (P4D1), IκBα (C-21) and Actin (I-19) were purchased from Santa Cruz Biotechnology. Antibodies against RIP1 were purchased from BD Biosciences. Antibodies against γ-tubulin (GTU-88) were purchased from Sigma-Aldrich. Antibodies against p52/p100 (4882), Phospho-IκBα (9246), Phospho-IKK (2697), Phospho-JNK (4671), JNK (9251), Phospho-ERK (4377), ERK (4695), Phospho-TAK1 (4536), TAK1 (4505), TNF (11948), and p65 (8242) were purchased from Cell Signaling Technology (Beverly, MA). Anti-rabbit Alexa Fluor 488 or 594 conjugate secondary antibodies (4412 or 8889) and anti-mouse Alexa Fluor 594 conjugate secondary antibody (8890) were from Cell Signaling Technology. OCT1 consensus oligonucleotide was purchased from Promega (Madison, WI).

Jackson S.S., Oberley C., Hooper C.P., Grindle K., Wuerzberger-Davis S., Wolff J., McCool K., Rui L, & Miyamoto S. (2014). Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling. Experimental cell research, 331(1), 58-72.

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Protein Expression and Analysis

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Cells were lysed in buffer containing 62.5 mmol/L Tris (pH 6.5), 5% glycerol, 2% SDS, 5% 2-mercaptoethanol, 1× protease inhibitor complete mixture (Roche Applied Science) and 1x phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Protein concentrations were determined using Bradford assay against a standard of bovine serum albumin. Lysate protein (25 μg) was resolved by electrophoresis on SDS-polyacrylamide gels (Mini-PROTEAN TGX Precast Gels, Bio-Rad) and transferred to Immobilon-P membranes (Millipore). Membranes were blocked in 5% milk. Primary antibodies were p65 (8242), EZH2 (5246), SUZ12 (3737) and Actin (3700) from Cell Signaling Technology, RelB (sc-226) from Santa Cruz Biotechnology, H3K27me3 (07–449) from Millipore. Membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Promega) and complexes were detected using the Supersignal West Pico Chemiluminescent Substrate Kit (Pierce).

Lawrence C.L, & Baldwin A.S. (2016). Non-Canonical EZH2 Transcriptionally Activates RelB in Triple Negative Breast Cancer. PLoS ONE, 11(10), e0165005.

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Protein Expression Analysis by Western Blot

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Western blot analysis was done as described previously^{50 (link)}. Antibodies were obtained from several providers. Cleaved caspase-3 (9661), Cleaved caspase-9 (7237), Caspase-3 (9662), Caspase-9 (9508) and p65 (8242) were from Cell Signaling (Danvers, MA, USA), β-actin (sc-130300), PCNA (sc-25280), Bax (sc-7480), p50 (sc-114), MMP-2 (sc-13595), MMP-3 (sc-6839), MMP-13 (sc-12363), normal mouse IgG (sc-2025) and MMP-9 (sc-127590 were purchased from Santacruz (Dallas, TX, USA) and IL-32 (513601) was obtained from BioLegend, (San Diego, CA USA). TIMP3 (ab39184) and DNMT1 (ab19905) were obtained from Abcam (Cambridge, MA, USA).

Yun J., Park M.H., Son D.J., Nam K.T., Moon D.B., Ju J.H., Hwang O.K., Choi J.S., Kim T.H., Jung Y.S., Hwang D.Y., Han S.B., Yoon D.Y, & Hong J.T. (2018). IL-32 gamma reduces lung tumor development through upregulation of TIMP-3 overexpression and hypomethylation. Cell Death & Disease, 9(3), 306.

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Antibody Characterization for NF-κB Pathway

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Antibodies for NF-κB p50 (13586, 1:1000), p65 (8242, 1:1000), IκBα (4814, 1:1000), p-p65 (S536) (3033, 1:1000), p-IκBα (Ser32/36) (9246, 1:1000), anti-TNF (6945, 1:1000), and His-Tag (2365, 1:2000) were obtained from Cell Signalling Technology. Anti-hNMI (ab183724, 1:1000), anti-HMGB1 (ab79823, 1:1000), anti-LDHB1 (ab75167, 1:1000), anti-LaminB (ab133741, 1:1000), and anti-N-myc (ab24193, 1:500) antibodies were obtained from Abcam. Anti-β-actin (A5441, 1:10000) antibody was obtained from Sigma-Aldrich. Polyclonal antibody for mNMI (Anti-mNMI, 1:1000) was prepared using recombinant mNMI protein according to previous report^{46 (link)}. Anti-hIFP35 (H00003430-D01P, 1:1000) antibody was obtained from Abnova. The ELISA kits for hNMI (CSB-EL015893HU), mNMI (CSB-EL015893MO), mouse AST (CSB-E12649m), and mouse ALT (CSB-E16539m) were obtained from CUSABIO. The ELISA kits for mIFP35 (E10460m) was purchased from Wuhan EIAab Science Co. The ELISA kits for mouse IL-6 (431305) and mouse TNF (430905) were purchased from Biolegend.
The activity and specificity of anti-hNMI (ab183724), anti-mNMI, anti-hIFP35 (H00003430-D01P), mNMI (CSB-EL015893MO), and mIFP35 (E10460m) ELISA kits were tested as shown in Supplementary Fig. 6.

Xiahou Z., Wang X., Shen J., Zhu X., Xu F., Hu R., Guo D., Li H., Tian Y., Liu Y, & Liang H. (2017). NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury. Nature Communications, 8, 950.

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Protein Expression and Antibody Validation

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Anti-β-actin (A1978), horseradish peroxidase (HRP)-anti-Flag (M2) (A8592), anti-mouse-IgG-HRP (AP308P), and anti-rabbit-IgG-HRP (AP132P) were purchased from Sigma (St. Louis, MO, USA). HRP-anti-hemagglutinin (12013819001) was purchased from Roche Applied Science (Switzerland, Basel). Anti-c-Myc (HT101) was purchased from TransGen Biotech (Beijing, China). Anti-TAK1 (5206) and p65 (8242) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LMNB1 (101237-T32) and anti-STUB1 (12496-R034) were purchased from Sino Biological (Beijing, China). Anti-CHIP (sc-133066) was purchased from Santa Cruz Biotechnology (San Diego, CA, USA). TNFα Recombinant Human Protein (10602HNAE25) was purchased from Thermo Fisher (Waltham, MA, USA).
Empty vector pcDNA3.1 was kindly provided by Dr Jun Cui (Sun Yat-sen University, Guangzhou, China). Target genes were cloned from SHSY5Y cDNA and then subcloned into the pcDNA3.1 vector. The recombinant plasmids were confirmed by DNA sequencing in Sangon Biotech (Shanghai, China).

Liu Y., Sun Y., Han S., Guo Y., Tian Q., Ma Q, & Zhang S. (2021). CHIP promotes the activation of NF-κB signaling through enhancing the K63-linked ubiquitination of TAK1. Cell Death Discovery, 7, 246.

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Cell Lysis and Protein Analysis

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Cells were lysed in RIPA lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 50 mM EDTA) with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific). Lysates were run on SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. Membranes were immunoblotted with anti-ICAM-1 BBIG-I1 (1:1,000), VCAM-1 BBIG-V1 (1:1,000), E-selectin BBIG-E4 (1:1,000) (R&D systems), MAP4K4 A301-503A (1:2,000) (Bethyl), VE-cadherin 2500 (1:2,000), p-JNK1/2 4668 (1:1,000), total JNK1/2 9258 (1:1,000), p-p38 MAPK 4631 (1:1,000), p-IκBα 9246 (1:1,000), IκBα 4814 (1:1,000), p-p65 3039 (1:1,000), p65 8242 (1:1,000), HGK 3485 (1:1,000), p-Erk 4370 (1:1,000) (Cell Signaling Technology), total p38 MAPK sc-535 (1:1,000), total Erk1 sc-93 (1:4,000) (Santa Cruz) or lamin-β1 ab16048 (1:1,000) (Abcam) antibodies. Uncropped western blot images are provided as Supplementary Figs 4–6.

Roth Flach R.J., Skoura A., Matevossian A., Danai L.V., Zheng W., Cortes C., Bhattacharya S.K., Aouadi M., Hagan N., Yawe J.C., Vangala P., Menendez L.G., Cooper M.P., Fitzgibbons T.P., Buckbinder L, & Czech M.P. (2015). Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis. Nature Communications, 6, 8995.

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