Log In Sign up
The largest database of trusted experimental protocols

Phospho nf κb p65 ser536 93h1

Manufactured by Cell Signaling Technology
Visit Supplier
Sourced in United States

Phospho-NF-κB p65 (Ser536) (93H1) is a rabbit monoclonal antibody that detects endogenous levels of NF-κB p65 protein only when phosphorylated at serine 536.

Automatically generated - may contain errors

Lab products found in correlation

Nf κb p65 c 20, by Santa Cruz Biotechnology (2 mentions) Phospho iκbα ser32 14d4, by Cell Signaling Technology (1 mentions) Annexin 5 apc apoptosis detection kit, by Keygen Biotech (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Rabbit β tubulin ab6046, by Abcam (1 mentions) Rabbit pcna abs, by Santa Cruz Biotechnology (1 mentions) P38 mapk, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Phospho iκbα ser32 36 5a5, by Cell Signaling Technology (1 mentions) Nf κb p50 h 119, by Santa Cruz Biotechnology (1 mentions) Mcl1 d35a5, by Cell Signaling Technology (1 mentions) Ac 15, by Merck Group (1 mentions) Flag m2, by Merck Group (1 mentions) Phospho jnk, by Cell Signaling Technology (1 mentions) Cd226, by BioLegend (1 mentions) Granzyme b, by BioLegend (1 mentions) Annexin 5, by BD (1 mentions) Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (1 mentions) Recombinant tnf α, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions)

10 protocols using phospho nf κb p65 ser536 93h1

1

Molecular Mechanisms of NF-κB Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols
The reagents and antibodies used in this study are α-Tubulin Rabbit Polyclinal Antibody (Beyotime, Shanghai, China, AF0001); rabbit monoclonal antibodies against phospho-NF-κB p65 (Ser536) (93H1) (Cell Signaling Technology, Danvers, MA, USA, 3033T); rabbit monoclonal antibodies against phospho-IκBα (Ser32) (14D4) (Cell Signaling Technology, Danvers, MA, USA, 2859T); IRDye 800CW Goat (polyclonal) Anti-Rabbit IgG (H + L), Highly Cross Adsorbed (LI-COR, 926-32211); IRDye 800CW Goat (polyclonal) Anti-Mouse IgG (H + L), Highly Cross Adsorbed (LI-COR, 926-32210); Dual-Luciferase Reporter Assay System, Promega, USA, E1910; Annexin V-APC apoptosis detection kit (KeyGEN BioTECH, Nanjing, China, KGA1021).
Gao Q., Yang Y., Quan W., Zheng J., Luo Y., Wang H., Chen X., Huang Z., Chen X., Xu R., Zhang G, & Gong L. (2021). The African Swine Fever Virus with MGF360 and MGF505 Deleted Reduces the Apoptosis of Porcine Alveolar Macrophages by Inhibiting the NF-κB Signaling Pathway and Interleukin-1β. Vaccines, 9(11), 1371.
+ Open protocol
+ Expand
2

Comprehensive Immunoblot Analysis Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
The following primary antibodies were used: mouse GAPDH (ab9484), rabbit β-tubulin (ab6046) from Abcam (Cambridge, UK); rabbit phospho-Akt Ser473 (D9E XP), phospho-p38 MAPK (T180/Y182), phospho-STAT1 (Tyr701) (58D6), phospho-TAK1 (T184/187) (90C7), phospho-JNK (81E11) (T183/Y185), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), IκBα (44D4), IRAK1 (D51G7), MyD88 (D80F5), Histone H3 (3H1), and phospho-NF-κB p65 (Ser536) (93H1) from Cell Signaling Technology (Danvers, MA, USA); rabbit PCNA Abs were from Santa Cruz Biotech (Santa Cruz, CA, USA); sheep IRF5 and IRAK4 were from MRC-PPU Reagents (University of Dundee, Dundee, UK); goat TIRAP polyclonal Abs were from Invitrogen (#PA5-18439, Waltham, MA, USA), and mouse STAT1 antibodies were from BD Biosciences (#610185, Wokingham, UK). Secondary antibodies (HRP-linked) were from DAKO Denmark A/S (Glostrup, Denmark).
Nilsen K.E., Skjesol A., Frengen Kojen J., Espevik T., Stenvik J, & Yurchenko M. (2022). TIRAP/Mal Positively Regulates TLR8-Mediated Signaling via IRF5 in Human Cells. Biomedicines, 10(7), 1476.
+ Open protocol
+ Expand
3

Evaluating Inflammation Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
To evaluate p38 and NF-κB phosphorylation, 1 × 107 BMDMs were treated with rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), or LPS (100 ng/mL) for 24 h. Then, the cells were lysed in a buffer containing 100 mM NaCl, 20 mM Tris (pH 7.6), 10 mM EDTA (pH 8), 0.5% SDS, and 1% Triton X-100 with protease inhibitor cocktail (Sigma-Aldrich) and incubated for 20 min on ice. Laemmli sample buffer was added to the lysates, and the samples were boiled for 10 min. Proteins were then separated by SDS-PAGE on 10% polyacrylamide resolving gels and transferred to nitrocellulose membranes. The primary antibodies used were: Phospho-NF-κB p65 (Ser536, 93H1, 1:1000; Cell Signaling Denvers, MA, USA; cat. number 3033), NF-κB p50 (E-10) mouse (1:500; Santa Cruz, Dallas, TX, USA; cat number sc-8414), phospho-p38 MAPK (Thr180/Tyr182, 28B10, 1:100; Cell Signaling; cat. number 9216), p38 MAPK (1:1000; Cell Signaling; cat. number 9212), and glyceraldehyde-3-phosphate dehydrogenase (1:2000; Trevigen, Gaithersburg, MD, USA; cat. number 2275).
Costa Mendonça-Natividade F., Duque Lopes C., Ricci-Azevedo R., Sardinha-Silva A., Figueiredo Pinzan C., Paiva Alegre-Maller A.C., L. Nohara L., B. Carneiro A., Panunto-Castelo A., C. Almeida I, & Roque-Barreira M.C. (2019). Receptor Heterodimerization and Co-Receptor Engagement in TLR2 Activation Induced by MIC1 and MIC4 from Toxoplasma gondii. International Journal of Molecular Sciences, 20(20), 5001.
+ Open protocol
+ Expand
4

Immunoblotting Antibodies for Cell Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Primary antibodies reactive to β-actin (AC-15; Sigma–Aldrich, St. Louis, MO, USA), FLAG (M2; Sigma–Aldrich, St. Louis, MO, USA), ICAM-1 (15.2; Leinco Technologies, St. Louis, MO, USA), FLIP (Dave-2; Alexis® Biochemicals, San Diego, CA, USA), IκBα (25; BD Biosciences, San Jose, CA, USA), Mcl-1 (D35A5; Cell Signaling Technology, Danvers, MA, USA), NF-κB p50 (H-119; Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65 (C-20; Santa Cruz Biotechnology, Dallas, TX, USA), Poly(ADP-ribose) polymerase (PARP) (C2-10; Trevigen, Gaithersburg, MD, USA), phospho-IκBα (Ser32/36) (5A5; Cell Signaling Technology, Danvers, MA, USA), and phospho-NF-κB p65 (Ser536) (93H1; Cell Signaling Technology, Danvers, MA, USA) were obtained.
Harada M., Morimoto K., Kondo T., Hiramatsu R., Okina Y., Muko R., Matsuda I, & Kataoka T. (2017). Quinacrine Inhibits ICAM-1 Transcription by Blocking DNA Binding of the NF-κB Subunit p65 and Sensitizes Human Lung Adenocarcinoma A549 Cells to TNF-α and the Fas Ligand. International Journal of Molecular Sciences, 18(12), 2603.
+ Open protocol
+ Expand
5

Comprehensive Immune Cell Profiling by FACS

Check if the same lab product or an alternative is used in the 5 most similar protocols
Fluorescence-activated cell sorting studies were performed using: FITC-, PE-, PE-Cy5, APC-Cy7, PE-Cy7, Pacific blue-, or APC-conjugated anti-human antibodies to: CD3 (clone#: HIT3a, 1:50), CD8 (SK1 or RPA-T8, 1:20), IL-17 (BL168, 1:20), IFNγ (4 S.B3, 1:10), IL-10 (JES3-19F1, 1:10), IL-4 (8D4-8, 1:10), FOXP3 (206D, 1:10), CD39 (A1, 1:20), CD73 (AD2, 1:20), CD28 (CD28.2, 1:20), CD45RA (HI100, 1:20), CD226 (TX25, 1:20), CXCR3 (G025H7, 1:20), Tim3 (F38-2E2, 1:20), CCR6 (G034E3, 1:20), Perforin (B-D48, 1:20), Granzyme B (GB11, 1:20) and CCR7 (g043h7, 1:20) from BioLegend (San Diego, CA, USA); CD39 (BU61, 5 μg ml−1) from Ancell Corporation (Bayport, MN, USA); Annexin V from BD Biosciences (1:20; Franklin Lakes, NJ, USA); phospho-JNK (Thr183/Tyr185; G9, 1:20) and phospho-NFκB p65 (Ser536; 93H1, 1:20) from Cell Signalling Technology (Danvers, MA, USA); and Isotype control antibodies from Ancell Corporation or eBioscience (San Diego, CA, USA).
Antibodies used for western blot included the following: phospho-PI3K p85 (Tyr458; #4228, 1:800), phospho-Akt (Ser473; #9271, 1:1,000), phospho-mTOR (Ser2448; #2971, 1:1,000), mTOR (#2972, 1:1,000), pJNK (Thr183/Tyr185; #9251, 1:1,000) and phospho-NFκB p65 (Ser536; #3031, 1:1,000), from Cell Signaling Technology (Danvers, MA, USA); NOX2 (ab31092, 1 μg ml−1) and β-actin (AC-15, #ab6276, 1:40,000) from Abcam (Cambridge, MA, USA).
Bai A., Moss A., Rothweiler S., Serena Longhi M., Wu Y., Junger W.G, & Robson S.C. (2015). NADH oxidase-dependent CD39 expression by CD8+ T cells modulates interferon gamma responses via generation of adenosine. Nature Communications, 6, 8819.
+ Open protocol
+ Expand
6

Measuring NF-κB Activation in 3D Cell Culture

Check if the same lab product or an alternative is used in the 5 most similar protocols
After culturing cells in gels for 1 day, recombinant TNFα (100 ng/ml; Peprotech) was added. At indicated time points, cells were rapidly retrieved from alginate hydrogels by adding 30 mM EDTA in DMEM with 1% bovine serum albumin (BSA) for 5 min on ice. During this process, dead cells were labeled with the LIVE/DEAD fixable violet dead cell stain (1:1000; Thermo Fisher Scientific). Cells were then washed out with DMEM with 1% BSA and fixed with 4% paraformaldehyde in HBSS at RT for 10 min. After washing two times with PBS/0.1% BSA, cells were stained with either phospho–NF-κB p65 (Ser536) (93H1) or total NF-κB p65 (D14E12) antibody (both from Cell Signaling Technology) at 1:100 dilution in the staining buffer (HBSS/0.1% saponin/0.1% BSA) at RT for 2 hours. They were then washed out once with the staining buffer and incubated with a secondary antibody (donkey Alexa 647 anti-rabbit, Thermo Fisher Scientific) for 30 min at RT, followed by washing out and resuspension in HBSS. Flow cytometry analysis was done using LSRFortessa (BD). The sample incubated with the isotype control [rabbit immunoglobulin G (IgG), DA1E, Cell Signaling Technology] was used as a negative control. Signals from live cell fractions were used for analysis.
Wong S.W., Lenzini S., Cooper M.H., Mooney D.J, & Shin J.W. (2020). Soft extracellular matrix enhances inflammatory activation of mesenchymal stromal cells to induce monocyte production and trafficking. Science Advances, 6(15), eaaw0158.
+ Open protocol
+ Expand
7

Antibodies for Western Blot and Immunofluorescence

Check if the same lab product or an alternative is used in the 5 most similar protocols
All antibodies used for Western blot and immunofluorescence were described in Table 4.

The antibodies were used in Western blot and immunofluorescence

AntibodySourceIdentifierDilution
Sirt3HUABIOR1511-31:750
ACOD1Abcamab2224111:1000
Acetylated-lysine antibodyCell Signaling Technology (CST)94411:1500
Anti-SOD2/MnSOD (acetyl K68)Abcamab1370371:1000
Integrin beta 7Proteintech11,328–1-AP1:1000
NF-κB p65HUABIOET1603-121:1000
Phospho-NF-κB p65 (Ser536) (93H1)Cell Signaling Technology (CST)3033S1:750
Phospho-DRP1 (Ser616)Cell Signaling Technology (CST)3455S1:750
AMPK alpha 1HUABIOET1608-401:1000
Phospho-AMPKα (Thr172) (D4D6D)Cell Signaling Technology (CST)2535S1:750
CCL2HUABIOHA5002671:1000
CCR2HUABIOET1611-651:1000
iNOSAbcamab1789451:1500
COX2/Cyclooxygenase 2HUABIOET1610-231:1500
NLRP3(NLRC3)HUABIOET1610-931:1500
IL-1β (D3U3E)Cell Signaling Technology (CST)12703S1:1000
Caspase-1AdipogeneAG-20B-0042-C1001:1000
Myeloperoxidase (MPO)Abcamab2086701:100
ASC(WB)AdipogeneAG-25B-0006PF-C1001:1000
ASC(IF)Santa Cruz BiotechSc-5144141:50
TubulinHUABIOET1602-41:1500
GAPDHHUABIOHA7211361:1500
Lv L., Jiang H., Song D., Zhou X., Chen F., Ren L., Xie Y, & Zeng M. (2023). Sirt3 improves monosodium urate crystal-induced inflammation by suppressing Acod1 expression. Arthritis Research & Therapy, 25, 121.
+ Open protocol
+ Expand
8

Immunoblotting Analysis of STING Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols
The RAW264.7
cells were seeded into a 6 cm plate with 5 × 105 cells/plate.
After 24 h, the RAW264.7 cells were treated with control (DMSO) or
10, 25, and 50 μM MSA-2 or MSA-2-Pt for 3 h. RAW264.7 cells
were collected and lysed by in RIPA Lysis Buffer (Beyotime) containing
phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail
(Roche) on ice for 30 min. Protein lysates were resolved by SDS-PAGE
and electrotransferred to a nitrocellulose membrane. β-Actin
(Cat: 66009-1-Ig, Proteintech, 1:5000) was the inner control. The
primary antibodies were Phospho-STING (Ser365) (D8F4W) (Cat: 72971,
Cell Signaling Technology, 1:1000), Phospho-TBK1/NAK (Ser172) (D52C2)
(Cat: 5483, Cell Signaling Technology, 1:1000), Phospho-NF-κB
p65 (Ser536) (93H1) (Cat: 3033, Cell Signaling Technology, 1:1000),
Phospho-IRF-3 (Ser396) (D6O1M) (Cat: 29047, Cell Signaling Technology,
1:1000), Rabbit mAb 29047, TMEM173/STING Polyclonal antibody (Cat:
19851-1-AP, Proteintech, 1:1000). The secondary antibodies (Cat: ab216777,
abcam, 1:5000; Cat: ab216776, abcam, 1:5000) were used. An imaging
system (Amersham Typhoon) was used to detect the signal.
Wang M., Cai Y., He T., Zhang Y., Yi L., Li W, & Zhou P. (2024). Antitumor Effect of Platinum-Modified STING Agonist MSA-2. ACS Omega, 9(2), 2650-2656.
+ Open protocol
+ Expand
9

Quantification of NF-κB Pathway Proteins

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Total cell lysates were prepared by homogenization in modified RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors, as previously described (Castaldo et al., 2019 (link)). The membranes were incubated overnight with the following antibodies: Phospho-NF-κB p65 (Ser536) (93H1) (1:1000 dilution; Cell Signaling, Danvers, MA, United States #3033), NFκB p65 (C-20) (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, United States #sc-372), IKKα/β and IKKγ (1:1000 dilution; Santa Cruz Biotechnology, #sc-7607 and #sc-8032, respectively), anti-ZNF224 (T3) (Cesaro et al., 2021b (link)) and anti-GAPDH (1:1000 dilution; Cell Signaling #2118). The blots were visualized using Clarity Western ECL Substrate Kit (Bio-Rad Laboratories) and immunoreactive bands were detected by autoradiography or by ChemiDoc XRS Image System (Bio-Rad Laboratories). Quantification of protein bands was obtained by densitometric analysis using the ImageJ software.
Catapano R., Sepe L., Toscano E., Paolella G., Chiurazzi F., Barbato S.P., Bruzzese D., Arianna R., Grosso M., Romano S., Romano M.F., Costanzo P, & Cesaro E. (2022). Biological relevance of ZNF224 expression in chronic lymphocytic leukemia and its implication IN NF-kB pathway regulation. Frontiers in Molecular Biosciences, 9, 1010984.
+ Open protocol
+ Expand
10

Evaluating Anti-inflammatory Effects of Kefir Extracts

Check if the same lab product or an alternative is used in the 5 most similar protocols
Caco-2 cells were treated for 24 h with an even mixture of L. kefir, L. kefiranofaciens, and L. kefirgranum EV (1 × 10 9 particles/mL). For positive control, cells cultured with EV in the presence of 5 µM budenoside (Sigma-Aldrich, St. Louis, MO) were used. To detect the NF-κB protein, cells were collected and lysed in radio immunoprecipitation assay buffer (50 µL). After centrifugation at 20,000 × g for 20 min at 4°C for removal of cell debris, the pellet was discarded. Cell lysate in radio immunoprecipitation assay buffer was mixed with NuPAGE LDS sample buffer (5×; Invitrogen, Carlsbad, CA) and heated at 95°C for 10 min. Equal amounts of protein (40 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride filters (Protein Sequencing Membrane). After blocking overnight at 4°C with 5% nonfat milk, the blots were probed with primary antibodies for overnight at 4°C with gentle shaking. Phospho-NF-κB p65 (Ser536; 93H1) and GAPDH (D16H11) were purchased from Cell Signaling (Danvers, MA). After incubation of the membrane with the appropriate peroxidase-conjugated secondary antibodies (Abcam) for 1 h at room temperature, the reactive protein signals were visualized and evaluated by using the enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA).
Seo M.K., Park E.J., Ko S.Y., Choi E.W, & Kim S. (2018). Therapeutic effects of kefir grain Lactobacillus-derived extracellular vesicles in mice with 2,4,6-trinitrobenzene sulfonic acid-induced inflammatory bowel disease. Journal of dairy science, 101(10).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!