Anti phosphor iκbα

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phosphor-IκBα is a laboratory reagent used to detect the phosphorylated form of the IκBα protein. IκBα is a key regulator of the NF-κB signaling pathway, and its phosphorylation is a critical step in the activation of this pathway.

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Anti-IκBα (274 citations) Phosphor-IκBα (12 citations)

6 protocols using anti phosphor iκbα

Comprehensive Immunoblotting Analysis of EBV Proteins

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The following antibodies were used: anti-DOK1 (ab8112, Abcam), anti-E2F1 (KH-95; Santa Cruz Biotechnology), anti- β-Actin C4 (MP Biomedicals), anti-LMP1 (S12), anti- phosphor IκBα (#9246, Cell Signaling Technology), anti-total IκBα (#9242, Cell Signaling Technology), mouse IgG, rabbit IgG (Santa Cruz Biotechnology), anti-p65 (#3034, Cell Signaling Technology), anti-H3K4me3, and anti-H3K27me3 (Epigentek), anti-EZH2 (AC22; Cell Signaling Technology), anti-pRB (4H1, Cell Signaling Technology), anti-DNMT1 (60B1220, Abnova), anti-EBNA1 (1EB12, Santa Cruz Biotechnology), anti-EBNA2 (Novocastra), anti-EBNA3A (Exalpha), anti-EBNA3C (ab16128, Abcam). Immunoblotting was performed as described previously [29] (link).

Siouda M., Frecha C., Accardi R., Yue J., Cuenin C., Gruffat H., Manet E., Herceg Z., Sylla B.S, & Tommasino M. (2014). Epstein-Barr Virus Down-Regulates Tumor Suppressor DOK1 Expression. PLoS Pathogens, 10(5), e1004125.

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Western Blot Analysis of Key Inflammatory Signaling Pathways

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Western-blot analysis was performed as previous described [20 (link)]. Different primary polyclonal anti-iNOS, anti-Arg1, anti-IκBα (inhibitor of NF-κB), anti-phosphor-IκBα, anti-p65, anti-phosphor-p65, anti-p38, anti- phosphor-p38 and anti-tubulin antibody were all purchased from cell signaling technology (CST, USA) and used in the present study. After a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse immunoglobulin (Ig) G antibody (1:10,000; SABC) incubated for 1 h at 37 °C, signals were detected by the enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).

Dou F., Chu X., Zhang B., Liang L., Lu G., Ding J, & Chen S. (2018). EriB targeted inhibition of microglia activity attenuates MPP+ induced DA neuron injury through the NF-κB signaling pathway. Molecular Brain, 11, 75.

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Western Blot Analysis of NF-κB Pathway

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Western blot was conducted according to a standard method previously described [44 (link)], using anti-URGCP (Sigma, Saint Louis, MI), anti-phosphor-IκBα, anti-IκBα, anti-phosphor-IKKβ, anti-IKKβ, anti-p65, anti-c-Jun or anti-P84 antibodies (Cell signaling, Danvers, MA). Blotted membranes were stripped and re-blotted with an anti-GAPDH or anti-α-tubulin monoclonal antibody (Sigma) as a loading control. The nuclear proteins were extracted using CelLytic NuCLEAR Extraction Kit (Sigma) according to the manufacturer's instructions.

Cai J., Li R., Xu X., Zhang L., Wu S., Yang T., Fang L., Wu J., Zhu X., Li M, & Huang Y. (2015). URGCP promotes non-small cell lung cancer invasiveness by activating the NF-κB-MMP-9 pathway. Oncotarget, 6(34), 36489-36504.

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Western Blot Analysis of Lung Tissue

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Lung tissue was lysed by using RIPA buffer (Thermo Fisher Scientific). Equal amounts of proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio‐Rad Laboratories). The membrane was blocked with 5% bovine serum albumin (Sigma‐Aldrich) for 90 min at room temperature and incubated with a primary antibody for 14 h at 4℃. The primary antibodies included anti–PPAR‐γ (Abcam), anti‐TGF‐β1 (ProteinTech), anti‐phosphor‐Smad3 (Cell Signaling Technology), anti‐Smad3 (Cell Signaling Technology), anti‐phosphor‐Stat6 (Cell Signaling Technology), anti‐Stat6 (Santa Cruz Biotechnology), anti‐phosphor‐Stat3 (Cell Signaling Technology), anti‐Stat3 (Santa Cruz Biotechnology), anti‐β‐tubulin (ProteinTech), anti‐NFκB P65 (Abcam), anti‐IκBα (Abcam), anti‐IL‐1β (Abcam), anti‐phosphor‐NFκB P65 (Cell Signaling Technology), anti‐phosphor‐IκBα (Cell Signaling Technology) and anti‐GAPDH (Abcam). And then, membranes were incubated with anti‐rabbit/mouse IgG (H+L) (Abcam) for 1 h at room temperature. Chemiluminescence measurement was performed by using Amersham Imager 680 (GE Healthcare Life Science).

Wang S., Chen Y., Hong W., Li B., Zhou Y, & Ran P. (2022). Chronic exposure to biomass ambient particulate matter triggers alveolar macrophage polarization and activation in the rat lung. Journal of Cellular and Molecular Medicine, 26(4), 1156-1168.

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Western Blot Antibody Validation

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The following antibodies were used for Western blotting: anti-URGCP, anti-β-actin, anti-β-tubulin and anti-GAPDH (Sigma), anti-CDK2, anti-CDK4, anti-CDK6, anti-Cyclin D1, anti-Cyclin E1, anti-c-myc, anti-p65, anti-phosphor-IκBα and anti-P84 (Cell signaling, Danvers, MA), anti-Cyclin A (Santa Cruz, CA, USA). Details are described in supplementary methods.

Hong L., Qing O., Ji Z., Chengqu Z., Ying C., Hao C., Minhui X, & Lunshan X. (2017). Downregulation of miR-16 via URGCP pathway contributes to glioma growth. Scientific Reports, 7, 13470.

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Antibody Validation for m6A Studies

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The following antibodies were used in this study: anti-m⁶A (Synaptic Systems, 202003 for m⁶A-seq; Abcam, ab208577 for m⁶A-IP qPCR), anti-YTHDF1 (Proteintech, 66745–1-lg), anti-TRAF6 (Abcam, ab181622), anti-TRAF3 (Proteintech, 18099-1-AP), anti-β-actin (HuaAn, R1207–1), anti-Flag (Sigma-Aldrich, F3165), anti-MYC (Abcam, ab32), anti-DDX60 (Abcam, ab139807), anti-MyD88 (Proteintech, 23230-1-AP), anti-phosphor-p65 (Cell Signaling Technology, 3033), anti-phosphor-IκBα (Cell Signaling Technology, 2859), anti-TRIF (Proteintech, 23288-1-AP), anti-p65 (Proteintech, 10745-1-AP), anti-IκBα (Proteintech, 10268-1-AP), anti-BCLAF1 (Proteintech, 26809-1-AP), anti-THRAP3 (Proteintech, 19744-1-AP), anti-TRAF3 (Proteintech, 18099-1-AP), anti-phospho-IKKα/β (Cell Signaling Technology, 2078), anti-IKKα (HuaAn, ER30911), anti-IKKβ (HuaAn, ER1706-13), anti-p38 (HuaAn, ET1602-26), phospho-p38 (Cell Signaling Technology, 9211), anti-phospho-JNK (HuaAn, RT1488), anti-JNK (HuaAn, RT1550), anti-ERK1/2 (Cell Signaling Technology, 9102), phospho-ERK1/2 (HuaAn, ET1610–13), normal rabbit IgG (Cell Signaling Technology, 2729) and normal mouse IgG (Cell Signaling Technology, 5946).

Zong X., Xiao X., Shen B., Jiang Q., Wang H., Lu Z., Wang F., Jin M., Min J., Wang F, & Wang Y. (2021). The N6-methyladenosine RNA-binding protein YTHDF1 modulates the translation of TRAF6 to mediate the intestinal immune response. Nucleic Acids Research, 49(10), 5537-5552.

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