Phospho-IKB alpha (Ser32/Ser36) Antibody - #AF2002

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

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MSDS
Data Sheet
COA
Protocols
WB Handbook
IHC Protocol
IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IHC 1:50-1:500, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

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.
Conjugate:
Unconjugated.
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:

P25963(IKBA_HUMAN) >>Visit HPA database.

Gene(ID):
NFKBIA(4792)
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). An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury. Bioactive Materials, 2022 (PubMed: 35845318) [IF=18.9]

Application: WB    Species: Mouse    Sample:

Fig. 2 Macrophage phenotype regulation based on RA@BSA@Cur NPs. (a) The schematic illustration of RA@BSA@Cur NPs regulated macrophage polarization under LPS. (b) The immunofluorescence and quantification results of RAW264.7 cultured different BSA-related NPs in LPS condition. CD86+ M1 macrophage (yellow arrow) and CD206+ M2 macrophage (white arrow), nuclei (DAPI: blue). Scale bar, 50 μm. (c&d) The flow cytometry analysis and quantification results of RAW264.7 (gated on F4/80+) cultured different BSA-related NPs in LPS condition. (e) The macrophages' M1 inhibition and M2 promotion regulated by RA@BSA@Cur NPs may be through the NF-κB pathway. (f) The inflammatory factors IL-6, TNF-α, and IL-4 changes after RA@BSA@Cur treatment. (n = 3 independent samples). Statistical differences were determined by using the Analysis of Variance (ANOVA) with Bonferroni's multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant; a.u. means arbitrary unit).
2). Tetrahedral Framework Nucleic Acids Based Small Interfering RNA Targeting Receptor for Advanced Glycation End Products for Diabetic Complications Treatment. ACS Nano, 2023 (PubMed: 37751401) [IF=17.1]
3). Opsonization Inveigles Macrophages Engulfing Carrier-Free Bilirubin/JPH203 Nanoparticles to Suppress Inflammation for Osteoarthritis Therapy. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2024 (PubMed: 38593402) [IF=15.1]

Application: WB    Species: Mouse    Sample: RAW 264.7 cells

Figure 4 Mechanism of IgG/BRJ regulated macrophage polarization. A) ROS levels in RAW 264.7 cells after different treatments were detected by the DCFH‐DA probe. Scale bar = 100 µm. B) DCF fluorescence quantitative analysis. C) Expression of p‐mTOR, mTOR, p‐IκBα, IκBα, p‐NF‐κB P65, and NF‐κB P65 in RAW 264.7 after LPS stimulation and treatment with different formulations. D–F) Quantification analysis of (C). Data are expressed as mean ± SD (n = 3), NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, compared to the LPS groups or as indicated.
4). Arsenic retention in erythrocytes and excessive erythrophagocytosis is related to low selenium status by impaired redox homeostasis. Redox Biology, 2022 (PubMed: 35500533) [IF=11.4]
5). 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 Biology, 2020 (PubMed: 32193146) [IF=11.4]

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.)
6). Clearance of apoptotic cells by mesenchymal stem cells contributes to immunosuppression via PGE2. EBioMedicine, 2019 (PubMed: 31248835) [IF=11.1]

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.
7). Opsonized nanoparticles target and regulate macrophage polarization for osteoarthritis therapy: A trapping strategy. Journal of Controlled Release, 2022 (PubMed: 35489544) [IF=10.8]
8). Hyperphosphorylated tau mediates neuronal death by inducing necroptosis and inflammation in Alzheimer’s disease. Journal of Neuroinflammation, 2022 (PubMed: 35971179) [IF=9.3]

Application: WB    Species: Mouse    Sample: HT22 cells

Fig. 4NF-κB is required for hyperphosphorylated tau-mediated cytokine induction. A HT22 cells were transfected with vector or TauP301S following treatment with DMSO or Nec-1 (30 μM) for 48 h, and the lysates were analyzed by western blotting using indicated antibodies. B Representative confocal images (left) and quantification (right) of p65 in HT22 cells transfected with vector or TauP301S following treatment with DMSO or Nec-1 (30 μM) for 48 h. Scale bars, 10 μm. C mRNA was extracted from HT22 cells transfected with vector or TauP301S following treatment with DMSO or TPCA1 (4 μM) and quantified to determine levels of indicated cytokines by qPCR. D Effect of NF-κB inhibitor on the chemotaxis of pTau-induced cytokines on BV2 cells was analyzed by transwell assays, Scale bars, 100 μm. E, F, I HT22 cells were transfected with vector or TauP301S following treatment with DMSO or TPCA1 (4 μM) or TPCA1 (4 μM) + Nec-1 (30 μM) for 48 h; E cell death was measured measuring LDH levels; F levels of the indicated cytokines were analyzed using qPCR; I lysates were analyzed by western blotting using indicated antibodies. G, J HT22 cells were transfected with vector or TauP301S following treatment with DMSO or QNZ (5 μM) or QNZ (5 μM) + Nec-1 (30 μM) for 48 h. G Cell death was evaluated by measuring LDH levels; J lysates were analyzed by western blotting using indicated antibodies. H mRNA was extracted from HT22 cells transfected with vector or TauP301S following treatment with DMSO or SP600125 (5 μM), PH797804 (5 μM), or C176 (2 μM), followed by quantification to determine levels of the indicated cytokines by qPCR. Data are presented as the mean ± standard error of the mean (SEM) of three experiments, statistical analysis was performed using two-tailed unpaired t test in E, G and one-way ANOVA with Dunnett’s multiple comparisons test in C, F. K NC HT22, RIPK1-KO HT22, RIPK3-KO HT22, and MLKL-KO HT22 cells were transfected with vector or TauP301S, and lysates were analyzed by western blotting using indicated antibodies. L Representative confocal images (left) and quantification (right) of p65 in NC HT22, RIPK1-KO HT22, RIPK3-KO HT22, and MLKL-KO HT22 cells transfected with vector or TauP301S. Scale bars, 10 μm. Data are presented as the mean ± standard error of the mean (SEM) of three experiments, and statistical analysis was performed using two-tailed unpaired t test in B, L
9). 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. PHARMACOLOGICAL RESEARCH, 2020 (PubMed: 31863867) [IF=9.3]

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.
10). Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice. npj Biofilms and Microbiomes, 2023 (PubMed: 36755021) [IF=9.2]
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