Iκbα c 21

Manufactured by Santa Cruz Biotechnology

Sourced in United States

IκBα (C-21) is a laboratory product offered by Santa Cruz Biotechnology. It is a protein that functions as an inhibitor of the NF-κB transcription factor. The product is intended for use in research applications.

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5 protocols using iκbα c 21

Cytoplasmic and Nuclear Extracts Analysis

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Cells were pretreated with or without of complex 5 (25 µg/ml or 50 µg/ml) for 4 h before treatment with LPS (10 ng/ml) or TNFα (10 ng/ml), for the indicated times. The cytoplasmic extracts were prepared by using the hypotonic lysis buffer (10 mM Tris, pH 7.5; 1.5 mM MgCl₂; 10 mM KCl; 0.5 mM DTT; 0.5 mM PMSF; 1× Protease Inhibitor, 0.1% NP40) followed by incubation on ice for 20 min and centrifugation at 3k rpm at 4°C for 10 min. The supernatants were collected as cytoplasmic extracts. The nuclear pellets were resuspended in the whole cell lysis buffer (25 mM Tris, pH7.5; 420 mM NaCl; 1.5 mM MgCl₂; 0.2 mM EDTA; 25% Glycerol; 0.5 mM DTT; 0.5 mM PMSF; 1× Protease Inhibitor). The nuclear extracts were then collected by centrifugation at 15 krpm at 4°C for 10 min. Immunoblotting was performed using antibodies against PARP (F-2, N-20), HSP90a (C-20), p65 (C-20), IκBα (C-21) and p-IκBα from Santa Cruz Biotech.

Shawish H.B., Wong W.Y., Wong Y.L., Loh S.W., Looi C.Y., Hassandarvish P., Phan A.Y., Wong W.F., Wang H., Paterson I.C., Ea C.K., Mustafa M.R, & Maah M.J. (2014). Nickel(II) Complex of Polyhydroxybenzaldehyde N4-Thiosemicarbazone Exhibits Anti-Inflammatory Activity by Inhibiting NF-κB Transactivation. PLoS ONE, 9(6), e100933.

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Integrin-Mediated Signaling Pathways

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The antibodies to β-actin, IκBα (C-21), and NF-κB p52 (K-27) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies to NF-κB p65 and integrin α2 (VLA-2α) were from Abcam (Cambridge, MA, USA) and BD Biosciences Pharmingen (San Diego, CA, USA), respectively. The antibodies to phospho-p38 (pp38, T180/Y182), p38, phospho-Akt (pAkt, T308), pAkt (S473), Akt, phospho-JNK (pJNK, T183/Y185), JNK, phospho-ERK (pERK, T202/Y204), and ERK were purchased from Cell Signaling Technology (CST; Beverly, MA, USA). The antibodies to active integrin β1 (AIIB2) were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA, USA). The human recombinant osteoprotegerin (OPG, TNFRSF11B)/Fc chimera and TNF-α were from R&D Systems (Minneapolis, MN, USA) and PeproTech (Rocky Hill, NJ, USA), respectively. The NF-κB inhibitor BAY-11-7082 and p38 mitogen-activated protein kinase inhibitor SB203580 were obtained from Calbiochem (San Diego, CA, USA) and CST, respectively.

Yamada T., Tsuda M., Wagatsuma T., Fujioka Y., Fujioka M., Satoh A.O., Horiuchi K., Nishide S., Nanbo A., Totsuka Y., Haga H., Tanaka S., Shindoh M, & Ohba Y. (2016). Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers. Scientific Reports, 6, 23545.

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IGF2 Imprinting and Regulation

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RNA was isolated from the cells or mouse prostate tissues using Rneasy Kit (Qiagen) with the addition of Dnase I to minimize DNA contamination. Imprinting was performed using a FluPE assay as previously described [6] (link). For human IGF2, a single nucleotide polymorphism identified on IGF2 exon 7 (G/C) was used to identify individual alleles. IGF2 imprinting was examined on Exon 6 (A/G) in mouse prostate tissues. Differences were determined by calculating the ratio of their respective spectral intensities [repressed allele (Ai)/active allele (Aa)]. Quantitative PCR was performed using an iCycler (Bio-Rad) and SYBR Green PCR master mix (Applied Biosystems) to measure gene expression, primers are available on request. Western blot was performed to detect protein expression using antibodies for CTCF (Cat #07-729, Millipore), NF-κB p50 (4D1, Santa Cruz), NF-κB p65 (c-20, Santa Cruz), NF-κB p100/52 (18D10, cell signaling), IKKα/β (sc-7607, Santa Cruz) and IκBα (c-21, Santa Cruz) and α-Tubulin (DM1A, EMD).

Yang B., Wagner J., Damaschke N., Yao T., Wuerzberger-Davis S.M., Lee M.H., Svaren J., Miyamoto S, & Jarrard D.F. (2014). A Novel Pathway Links Oxidative Stress to Loss of Insulin Growth Factor-2 (IGF2) Imprinting through NF-κB Activation. PLoS ONE, 9(2), e88052.

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Western Blot Analysis of Lung Proteins

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Lung protein extracts were prepared using the Nuclear Extract Kit (BestBio, China) following the manufacturer's instruction. Equal amounts of protein were separated by SDS-PAGE and subsequently blotted on polyvinylidene fluoride membranes (220 mA, 65 min). Blots were blocked in TBS solution containing 0.1% Tween 20 and 5% nonfat dry milk overnight at 4°C. The following antibodies and dilutions were used: TLR7 (Catalog number 2633, Cell Signaling Technology; 1 : 1000); MyD88 (D80F5) (Catalog number 4283, Cell Signaling Technology; 1 : 1000); TRAF6 (Catalog number 04-451, Millipore, USA; 1 : 1000); IκB-α (c-21) (sc-371, Santa Cruz Biotechnology, 1 : 200); p-IKKα/β (Ser 176) (sc-21661, Santa Cruz Biotechnology, 1 : 200); NF-κB p65 (sc-109, Santa Cruz Biotechnology, 1 : 200); p-NF-κB p65 (Ser 276) (sc-101749, Santa Cruz Biotechnology, 1 : 200); and GAPDH (14C10) (Catalog number 2118, Cell Signaling Technology; 1 : 1000). The membranes were blotted with appropriate secondary antibodies (Immunology Consultants Laboratory, Inc., USA; 1 : 5000), and the blotted proteins were visualized by enhanced chemiluminescence using a commercially available kit (Millipore, USA).

Chen L., Yan X., Yan Q., Fan J., Huang H., Shi X., Han L., Han T, & Zhu H. (2015). The Modified JiuWei QiangHuo Decoction Alleviated Severe Lung Injury Induced by H1N1 Influenza Virus by Regulating the NF-κB Pathway in Mice. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 790739.

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Protein Expression and Phosphorylation Analysis

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The antibodies and reagents were purchased from the following sources: NCOA3 (F-2) (Santa Cruz, sc5305), GAPDH (0411) (Santa Cruz, sc47724), COL2A1 (M2139) (Santa Cruz, sc52658), Aggrecan (6-B-4) (abcam, ab3778), COL1A1 (3G3) (Santa Cruz, sc293182), p65 (abcam, ab16502), phospho-p65 (S536) (abcam, ab28856), IκBα (C-21) (Santa Cruz, sc371), phospho-IκBα (Ser32/36) (Cell Signaling, #9246), Goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004), Goat anti-mouse IgG-HRP (Santa Cruz, sc-2302). 5-Azacytidine (Sigma, A2385), Bufalin (Sigma, B0261), BrdU (5-Bromo-2'-deoxyuridine) (Millipore, #19-160), ML120B (Sigma, SML1174), recombinant IL-1β (R&D System, NP-000567).

Zhang B., Li Z., Wang W., Guo J., Kang S., Liu S., Li H., Wang D, & Qi X. (2018). NCOA3 Loss Disrupts Molecular Signature of Chondrocytes and Promotes Posttraumatic Osteoarthritis Progression. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 49(6).

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