Product: Phospho-IKB alpha (Ser32/Ser36) Antibody
Catalog: AF2002
Source: Rabbit
Application: WB, IHC, IF/ICC, ELISA(peptide)
Reactivity: Human, Mouse, Rat, Monkey
Prediction: Pig, Bovine, Sheep, Rabbit, Dog, Chicken
Mol.Wt.: 39kD; 36kD(Calculated).
Uniprot: P25963
RRID: AB_2834433

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Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:500, IF/ICC 1:100-1:500, ELISA(peptide) 1:20000-1:40000
*The optimal dilutions should be determined by the end user.
Reactivity:
Human,Mouse,Rat,Monkey
Prediction:
Pig(100%), Bovine(100%), Sheep(100%), Rabbit(100%), Dog(100%), Chicken(92%)
Clonality:
Polyclonal
Specificity:
Phospho-IKB alpha (Ser32/Ser36) Antibody detects endogenous levels of IKB alpha only when phosphorylated at Serine 32/Serine 36.
RRID:
AB_2834433
Cite Format: Affinity Biosciences Cat# AF2002, RRID:AB_2834433.
Purification:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

I kappa B alpha; I-kappa-B-alpha; IkappaBalpha; IkB-alpha; IKBA; IKBA_HUMAN; IKBalpha; MAD 3; MAD3; Major histocompatibility complex enhancer-binding protein MAD3; NF kappa B inhibitor alpha; NF-kappa-B inhibitor alpha; NFKBI; NFKBIA; Nuclear factor of kappa light chain gene enhancer in B cells; Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha;

Immunogens

Immunogen:
Uniprot:
Gene(ID):
Description:
NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA (MIM 164014), or RELB (MIM 604758) to form the NFKB complex. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm.
Sequence:
MFQAAERPQEWAMEGPRDGLKKERLLDDRHDSGLDSMKDEEYEQMVKELQEIRLEPQEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLHLAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLASVGVLTQSCTTPHLHSILKATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLLLKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL

Predictions

Predictions:

Score>80(red) has high confidence and is suggested to be used for WB detection. *The prediction model is mainly based on the alignment of immunogen sequences, the results are for reference only, not as the basis of quality assurance.

Species
Results
Score
Pig
100
Bovine
100
Sheep
100
Dog
100
Rabbit
100
Chicken
92
Xenopus
69
Horse
0
Zebrafish
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

PTMs - P25963 As Substrate

Site PTM Type Enzyme
Ubiquitination
K21 Sumoylation
K21 Ubiquitination
K22 Sumoylation
K22 Ubiquitination
S32 Phosphorylation P68400 (CSNK2A1) , O14965 (AURKA) , Q99558 (MAP3K14) , Q15418 (RPS6KA1) , Q14164 (IKBKE) , O00141 (SGK1) , P19525 (EIF2AK2) , P43250 (GRK6) , Q96KB5 (PBK) , O15111 (CHUK) , O14920 (IKBKB) , P51812 (RPS6KA3) , P34947 (GRK5) , Q9Y6K9 (IKBKG) , Q15349 (RPS6KA2)
S36 Phosphorylation P68400 (CSNK2A1) , Q99558 (MAP3K14) , Q9UHD2 (TBK1) , P43250 (GRK6) , O15111 (CHUK) , Q15418 (RPS6KA1) , O14920 (IKBKB) , Q14164 (IKBKE) , O14965 (AURKA)
K38 Ubiquitination
Y42 Phosphorylation P12931 (SRC) , P06213 (INSR) , P06239 (LCK) , P43405 (SYK)
K47 Ubiquitination
K67 Ubiquitination
K87 Ubiquitination
T90 Phosphorylation
K98 Ubiquitination
S166 Phosphorylation
K238 Ubiquitination
T273 Phosphorylation
S283 Phosphorylation P68400 (CSNK2A1)
S288 Phosphorylation P68400 (CSNK2A1)
T291 Phosphorylation P68400 (CSNK2A1)
S293 Phosphorylation P68400 (CSNK2A1)
T299 Phosphorylation P68400 (CSNK2A1)
Y305 Phosphorylation P00519 (ABL1) , A0A173G4P4 (Abl fusion)

Research Backgrounds

Function:

Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.

PTMs:

Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. Phosphorylation at positions 32 and 36 is prerequisite to recognition by UBE2D3 leading to polyubiquitination and subsequent degradation.

Sumoylated; sumoylation requires the presence of the nuclear import signal. Sumoylation blocks ubiquitination and proteasome-mediated degradation of the protein thereby increasing the protein stability.

Monoubiquitinated at Lys-21 and/or Lys-22 by UBE2D3. Ubiquitin chain elongation is then performed by CDC34 in cooperation with the SCF(FBXW11) E3 ligase complex, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. The resulting polyubiquitination leads to protein degradation. Also ubiquitinated by SCF(BTRC) following stimulus-dependent phosphorylation at Ser-32 and Ser-36.

Deubiquitinated by porcine reproductive and respiratory syndrome virus Nsp2 protein, which thereby interferes with NFKBIA degradation and impairs subsequent NF-kappa-B activation.

Subcellular Location:

Cytoplasm. Nucleus.
Note: Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Subunit Structure:

Interacts with RELA; the interaction requires the nuclear import signal. Interacts with NKIRAS1 and NKIRAS2. Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, ELP1 and MAP3K14. Interacts with isoform 1 and isoform 2 of RWDD3; the interaction enhances sumoylation. Interacts (when phosphorylated at the 2 serine residues in the destruction motif D-S-G-X(2,3,4)-S) with BTRC. Associates with the SCF(BTRC) complex, composed of SKP1, CUL1 and BTRC; the association is mediated via interaction with BTRC. Part of a SCF(BTRC)-like complex lacking CUL1, which is associated with RELA; RELA interacts directly with NFKBIA. Interacts with PRMT2. Interacts with PRKACA in platelets; this interaction is disrupted by thrombin and collagen. Interacts with HIF1AN. Interacts with MEFV. Interacts with DDRGK1; positively regulates NFKBIA phosphorylation and degradation.

(Microbial infection) Interacts with HBV protein X.

Family&Domains:

Belongs to the NF-kappa-B inhibitor family.

Research Fields

· Cellular Processes > Cell growth and death > Apoptosis.   (View pathway)

· Environmental Information Processing > Signal transduction > cAMP signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > NF-kappa B signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > TNF signaling pathway.   (View pathway)

· Human Diseases > Endocrine and metabolic diseases > Insulin resistance.

· Human Diseases > Infectious diseases: Bacterial > Epithelial cell signaling in Helicobacter pylori infection.

· Human Diseases > Infectious diseases: Bacterial > Shigellosis.

· Human Diseases > Infectious diseases: Bacterial > Legionellosis.

· Human Diseases > Infectious diseases: Parasitic > Leishmaniasis.

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Parasitic > Toxoplasmosis.

· Human Diseases > Infectious diseases: Viral > Hepatitis C.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Measles.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Human Diseases > Infectious diseases: Viral > HTLV-I infection.

· Human Diseases > Infectious diseases: Viral > Herpes simplex infection.

· Human Diseases > Infectious diseases: Viral > Epstein-Barr virus infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Viral carcinogenesis.

· Human Diseases > Cancers: Specific types > Prostate cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Small cell lung cancer.   (View pathway)

· Organismal Systems > Immune system > Chemokine signaling pathway.   (View pathway)

· Organismal Systems > Development > Osteoclast differentiation.   (View pathway)

· Organismal Systems > Immune system > Toll-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > RIG-I-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Cytosolic DNA-sensing pathway.   (View pathway)

· Organismal Systems > Immune system > IL-17 signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Th1 and Th2 cell differentiation.   (View pathway)

· Organismal Systems > Immune system > Th17 cell differentiation.   (View pathway)

· Organismal Systems > Immune system > T cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > B cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Nervous system > Neurotrophin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Adipocytokine signaling pathway.

· Organismal Systems > Endocrine system > Relaxin signaling pathway.

References

1). Zhang Z et al. Clearance of apoptotic cells by mesenchymal stem cells contributes to immunosuppression via PGE2. EBioMedicine 2019 Jun 24 (PubMed: 31248835) [IF=11.205]

Application: WB    Species: mouse    Sample: MSCs

Fig. 5. |PGE2 Production in AC-MSCs is Dependent on NF-κB Signalling. (a) MSCs were cocultured with ACs at a ratio of 1:20 for the indicated time. The expressions of IκBα, p65 and their phosphorylated forms in MSCs were analysed by western blot.

2). Wu H et al. Breaking the vicious loop between inflammation, oxidative stress and coagulation, a novel anti-thrombus insight of nattokinase by inhibiting LPS-induced inflammation and oxidative stress. Redox Biol 2020 Mar 11;32:101500 (PubMed: 32193146) [IF=10.787]

Application: WB    Species: Mice    Sample: RAW264.7 cells

Fig. 3. NK inhibited LPS-induced NF-κB activation in RAW264.7 cells. (A) Effect of NK on LPS-induced IκBα activation in RAW264.7 cells. Cells were pretreated with NK (0.08, 0.15, and 0.30 FU/mL) for 1 h before being exposed to LPS (0.1 μg/ml) for 6 h. Equal amounts of total cell lysates were loaded and subjected to immunoblot analysis. β-actin was used as the control for equal protein loading and protein integrity. **P < 0.01, vs. Control; #P < 0.05, ##P < 0.01, vs. LPSstimulated cells. (B) Effect of NK on LPS-induced P65 nuclear-translation in RAW264.7 cells. Cells were pretreated with NK (0.30 FU/mL) for 1 h and then exposed to LPS (0.1 μg/mL) for 6 h. Immunofluorescence analysis was performed. P65 protein was marked with green fluorescent, and the nucleus were dyed blue with Hochest. Scale bar: 40 μm. (C) Effect of NK on LPS-induced TNF-α release in RAW264.7 cells. Cells were pretreated with NK (0.08, 0.15, 0.30 FU/mL) for 1 h before LPS (0.1 μg/mL) stimulation for 24 h. Cell supernatant was collected to detect TNF-α level by ELISA. (D) Effect of NK on LPS-induced IL-6 release in RAW264.7 cells. Cells were pretreated with NK (0.08, 0.15, 0.30 FU/mL) for 1 h before LPS (0.1 μg/mL) stimulation for 24 h. Cell supernatant was collected to detect IL-6 level by ELISA. **p < 0.01, ***p < 0.001, vs. control; #p < 0.05, ##p < 0.01, ###p < 0.001, vs. LPS-stimulated cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3). Li C et al. Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: Impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-κB pathway. Pharmacol Res 2020 Feb;152:104603 (PubMed: 31863867) [IF=10.334]

Application: WB    Species: Mice    Sample: colonic tissues

Fig. 6. Effect of OBB on the activation of TLR4-MyD88-NF-κB signaling pathway in DSS-induced colonic tissues. (A) Representative Western blotting images of TLR4, MyD88, cytoplasmic p65, nuclear p65, p-IκBα and IκBα. Changes in the relative protein expression levels of TLR4 (B), MyD88 (C), nuclear p65 (D), cytoplasmic p65 (E), and p-IκBα/IκBα ratio (F) were measured. Data are shown as the mean ± SEM (n = 3). # P < 0.05, ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group.

4). Meng DF et al. S100A14 suppresses metastasis of nasopharyngeal carcinoma by inhibition of NF-kB signaling through degradation of IRAK1. Oncogene 2020 Jun 17. (PubMed: 32555330) [IF=8.756]

Application: WB    Species: Human    Sample: NPC cells

Fig 4.b NF-KB signaling makers in S100A14 overexpressing cells and S100A14 knocked-down cells were evaluated by immunoblotting.

5). Wu J et al. Patchouli alcohol attenuates 5-fluorouracil-induced intestinal mucositis via TLR2/MyD88/NF-kB pathway and regulation of microbiota. Biomed Pharmacother 2020 Jan 28;124:109883 (PubMed: 32004938) [IF=7.419]

Application: WB    Species: rat    Sample:

Fig. 3.| Effect of PA on inflammtory cytokines (n = 8) and TLR2/MyD88/NF-κB pathway proteins (n = 3). (a–e) Levels of TNF-α, IL-1β, IL-6, IL-10, and MPO; (f–h)Expressions of TLR2 and MyD88 proteins; (i–j) Expressions of IκBα and p-IκBα proteins; (k–l) Expression of nuclear NF-κB p65 protein (400 ×). Values were represented the mean ± SEM. **P < 0.01, *P < 0.05 versus 5-FU group and ##P < 0.01 versus normal group.

6). Liu J et al. Investigating the Mechanisms of Jieduquyuziyin Prescription Improves Lupus Nephritis and Fibrosis via FXR in MRL/lpr Mice. Oxid Med Cell Longev 2022 Jul 9;2022:4301033. (PubMed: 35855861) [IF=7.310]

7). Han B et al. Down-regulation of lncRNA DNAJC3-AS1 inhibits colon cancer via regulating miR-214-3p/LIVIN axis. Bioengineered 2020 Dec;11(1):524-535. (PubMed: 32352854) [IF=6.832]

Application: WB    Species: Human    Sample: LoVo cells

Figure 3. DNAJC3-AS1 affected miR-214-3p and its related signaling pathway. (a) Putative miR-214-3p binding and mutated sites in DNAJC3-AS1. (b) Dual-luciferase reporter assay was performed to determine the luciferase activity of 293 T cells. (c) The expression of miR-214-3p was evaluated after the down-regulation of DNAJC3-AS1 in LoVo cells. (d-f) The protein levels of LIVIN (d), p-IκB (e), IκB (e), p-P65 (f) and P65 (f) in LoVo cells were detected by western blot. Data is shown as mean ± SD, ** p < 0.01, *** p < 0.001.

8). Li X et al. Down-regulation of ROCK2 alleviates ethanol-induced cerebral nerve injury partly by the suppression of the NF-κB signaling pathway. Bioengineered 2020 Dec;11(1):779-790. (PubMed: 32684089) [IF=6.832]

Application: WB    Species: Rat    Sample:

Figure 5. Down-regulation of ROCK2 suppresses the NF-κB signaling pathway induced by ethanol in rats. a. The expression of p-IκB, IκB was determined by Western blot.b. The expression of nuclear NF-κB p65 and cytoplasmic NF-κB p65 was determined by Western blot.c. The activation of NF-κB was measured by EMSA.Data are expressed as mean± SD. ##p < 0.01 compared to Control group; **p < 0.01 compared to Ethanol+Lv-control group.

9). Li CL et al. Comparison of anti-inflammatory effects of berberine, and its natural oxidative and reduced derivatives from Rhizoma Coptidis in vitro and in vivo. Phytomedicine 2019 Jan;52:272-283 (PubMed: 30599908) [IF=6.656]

10). Yuan L et al. Upregulation of UGT1A1 Expression by Ursolic Acid and Oleanolic Acid via the Inhibition of the PKC/NF-κB Signaling Pathway. Phytomedicine 2021 Aug 25;92:153726. (PubMed: 34536821) [IF=6.656]

Application: WB    Species: Human    Sample: HepG2 cells and Huh7 cells

Fig 2. Effects of PMA and LPS on UGT1A1 expression and NF-κB activity in HepG2 cells and Huh7 cells. HepG2 cells and Huh7 cells, respectively, were treated with PMA (100 nM) and/or LPS (10 μg/ml) for 24 h in the presence or absence of PDTC (20 μM). DMSO (0.1%) was used as the negative control. (A) Relative mRNA and (B) protein levels of UGT1A1 were quantified by qRT-PCR and western blot analyses, respectively. The experiments were normalized to GAPDH and compared to the negative control group. (C) Protein (cytoplasmic, nuclear or total) levels of p65, phospho-p65, IκBα, and phospho-IκBα were quantified by western blot analysis. Total-p65 was normalized to GAPDH, whereas the nucleus and cytoplasmic proteins were normalized to Lamin B1 and β-actin, respectively. All values were expressed as the mean ± S.E. of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001).

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