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Anti iκbα 44d4

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Anti-IκBα (44D4) is a mouse monoclonal antibody that recognizes the NF-κB inhibitor IκBα. It is designed for use in applications such as Western blotting to detect and quantify IκBα protein levels.

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Lab products found in correlation

Anti phospho iκbα ser32 36 5a5, by Cell Signaling Technology (3 mentions) Anti nf κb p65 d14e12, by Cell Signaling Technology (2 mentions) Image station 440, by Kodak (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Anti actin 1 19, by Santa Cruz Biotechnology (2 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Santa Cruz Biotechnology (2 mentions) Protoglow ecl, by National Diagnostics (2 mentions) Anti phospho iκbα 5a5, by Cell Signaling Technology (2 mentions) Anti vinculin, by Merck Group (2 mentions) Anti ubiquitin p4d1, by Santa Cruz Biotechnology (2 mentions) Anti hoil 1l clone 2e2, by Merck Group (2 mentions) Anti hoip, by Merck Group (2 mentions) Sab2102031, by Cell Signaling Technology (2 mentions) Anti sharpin, by Novus Biologicals (2 mentions) Anti phospho iκbα ser32, by Cell Signaling Technology (2 mentions) Myd88 d80f5, by Cell Signaling Technology (1 mentions) Anti phospho ikkα β ser176 180 16a6, by Cell Signaling Technology (1 mentions) Anti phospho nf κb p65 ser536 93h1, by Cell Signaling Technology (1 mentions) Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Anti-IκBα (274 citations) IκBα (44D4)) (4 citations)

7 protocols using anti iκbα 44d4

1

Western Blotting of NF-κB Pathway

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Western blotting was performed as previously described (Chen et al., 2002 (link); Xiao et al., 2019 (link)). Antibodies anti-myeloid differentiation primary response gene 88 (MyD88) (D80F5; #4283), anti-phospho-IKKα/β (Ser176/180) (16A6; #2697), anti-IKKα (#2682), Phospho-IKKα/β (Ser176/180) (16A6, #2697), anti-IκBα (44D4,#4812), anti-Phospho-IκBα (Ser32/36) (5A5, #9246), anti-NF-κB p65 (D14E12, #8242) and anti-Phospho-NF-κB p65 (Ser536) (93H1, #3033) were purchased from Cell Signaling Technology.
Su J., Guo K., Huang M., Liu Y., Zhang J., Sun L., Li D., Pang K.L., Wang G., Chen L., Liu Z., Chen Y., Chen Q, & Huang L. (2019). Fucoxanthin, a Marine Xanthophyll Isolated From Conticribra weissflogii ND-8: Preventive Anti-Inflammatory Effect in a Mouse Model of Sepsis. Frontiers in Pharmacology, 10, 906.
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2

Immunoblot Analysis of IκBα Phosphorylation

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Cells were lysed with cell lysis buffer (cell signaling) in the presence of protease inhibitor cocktail (complete, Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Santa Cruz Biotechnologies). Cell lysates were subjected to 10% SDS-PAGE, transferred to PVDF membranes (Bio-Rad) and were treated with primary and secondary antibodies, respectively. The blots were visualized using the protoglow ECL (National Diagnostics) and image station 440 (Kodak). Antibodies used were as follows: anti- IκBα (44D4; Cell Signaling), anti-phospho- IκBα (5A5; Cell Signaling), anti-actin (I-19; Santa Cruz Biotechnologies), HRP-conjugated anti-mouse IgG (Cell Signaling), HRP-conjugated anti-rabbit IgG (Cell Signaling), and HRP-conjugated anti-goat IgG (Santa Cruz Biotechnologies).
Nakagawa M.M., Chen H, & Rathinam C.V. (2018). Constitutive Activation of NF-κB Pathway in Hematopoietic Stem Cells Causes Loss of Quiescence and Deregulated Transcription Factor Networks. Frontiers in Cell and Developmental Biology, 6, 143.
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3

Immunoblot Analysis of Cellular Proteins

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For immunoblot analyses, cells were lysed with cell lysis buffer (Cell Signaling Technology) with protease inhibitor cocktail (Complete; Roche) and 1 mM PMSF (Santa Cruz Biotechnology, Inc.). Cell lysates were boiled with sample buffer (NuPAGE; Life Technologies) containing 1% 2-Mercaptoethanol (Sigma-Aldrich). Proteins were subjected to 8–12% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were blocked with either 5% bovine serum albumin (Life Technologies) or 5% skim milk and then treated with primary and secondary antibodies, respectively. The blots were visualized using the ProtoGlow ECL (National Diagnostics) and Image station 440 (Kodak). Antibodies used were as follows: anti-A20 (D13H3; Cell Signaling Technology), anti-IκBα (44D4; Cell Signaling Technology), anti–phospho-IκBα (5A5; Cell Signaling Technology), anti-actin (I-19; Santa Cruz Biotechnology, Inc.), HRP-conjugated anti–mouse and anti–rabbit IgG (Cell Signaling Technology), and HRP-conjugated anti–goat IgG (Santa Cruz Biotechnology, Inc.).
Nakagawa M.M., Thummar K., Mandelbaum J., Pasqualucci L, & Rathinam C.V. (2015). Lack of the ubiquitin-editing enzyme A20 results in loss of hematopoietic stem cell quiescence. The Journal of Experimental Medicine, 212(2), 203-216.
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4

Antibody Dilution Protocol for Western Blot

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The following antibodies were used in this study: anti-HOIL-1L (clone 2E2; Merck MABC576), anti-HOIP (Merck SAB2102031), anti-IκBα (44D4; Cell Signaling Technology 4812), anti-Ser32/36-phospho-IκBα (5A5; Cell Signaling Technology 9246), anti-SHARPIN (NOVUS Biologicals NBP2-04116), anti-ubiquitin (P4D1; Santa Cruz Biotechnology sc-8017), anti-vinculin (Merck V9131). All antibodies were diluted in TBS 5% (w/v) BSA, 0.05% (v/v) Triton according to the manufacturer’s recommended dilutions.
Rodriguez Carvajal A., Grishkovskaya I., Gomez Diaz C., Vogel A., Sonn-Segev A., Kushwah M.S., Schodl K., Deszcz L., Orban-Nemeth Z., Sakamoto S., Mechtler K., Kukura P., Clausen T., Haselbach D, & Ikeda F. (2021). The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains. eLife, 10, e60660.
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5

Immunoblot Analysis of Signaling Proteins

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Extracted proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Amersham, catalog no. 10600002), and incubated with the primary antibody for overnight at 4°C. Immunoblots were probed with the first antibody with anti-phospho-IκBα (Ser32) (#2859, Cell Signaling Technology), anti-IκBα (44D4) (#4814, Cell Signaling Technology), anti-phospho-p65 (Ser536) (#3033, Cell Signaling Technology), anti-p65 (D14E12) (#8242, Cell Signaling Technology), anti-ACE2 (#ab108209, Abcam) and β-actin (#CW0096M, CWBiotech). And ECL luminescent solution was used for detection.
Yan M., Dong Y., Bo X., Cheng Y, & Cheng J. (2021). Large Screening Identifies ACE2 Positively Correlates With NF-κB Signaling Activity and Targeting NF-κB Signaling Drugs Suppress ACE2 Levels. Frontiers in Pharmacology, 12, 771555.
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6

Antibody Panel for NF-κB Signaling

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The following antibodies were used in this study: anti-HOIL-1L (clone 2E2;
Merck MABC576), anti-HOIP (Merck SAB2102031), anti-IκBα (44D4; Cell Signalling Technology 4812), anti-Ser32/36-phospho-IκBα (5A5; Cell Signaling Technology 9246), anti-SHARPIN (NOVUS Biologicals NBP2-04116), anti-ubiquitin (P4D1; Santa Cruz Biotechnology sc-8017), anti-vinculin (Merck V9131). All antibodies were diluted in TBS 5% (w/v) BSA, 0.05% (v/v)
Triton according to the manufacturer's recommended dilutions.
Carvajal A.R., Gómez-Díaz C., Vogel A., Sonn-Segev A., Schodl K., Deszcz L., Orbán-Németh Z., Sakamoto S., Mechtler K., Kukura P., Clausen T., Haselbach D., & Ikeda F. (2020). The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains.
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7

Cellular Signaling Pathway Regulation Analysis

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Reagents and antibodies BA (purity≥98%) was purchased from Psaitong (Beijing, China). Dimethyl sulfoxide (DMSO) and LPS was obtained from Sigma (St. Louis, MO, USA). Antibodies used for western blotting in the present study were as follows: the primary antibodies against inducible Nitric Oxide Synthase (iNOS) (#13120), anticyclooxygenase-2 (COX-2) (#12282), anti-phospho-IKKα/β (S176/180) (16A6) (#2697P), anti-phospho-IκBα (Ser32) (#2859), anti-IκBα (44D4) (#4814), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101), anti-ERK1/2 (#9102), anti-phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-p38 MAPK (#9212), antiphospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-JNK2 (56G8) (#9258), anti-NF-κB p65 (D14E12) ( #8242) were both purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IKKα (CHUK) (#A2062) was from ABclonal Technology (Wuhan, HB, China), β-tubulin (#CW0098A) and GAPDH (#CW0266A) were from CWBiotech (Beijing, China). Anti-Histone H2B were obtained from Santa Cruz Biotechnology Co., Ltd. (Shanghai, China).
Liu S., Yan M., Liao Y., & Cheng Y. (2021). Bullatine a Exerted Anti-Inflammatory Effects by Inhibiting JNK/ROS/NF-κB Pathway and Attenuates Systemic Inflammatory Response in LPS-Challenged Mice.
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