Rabbit anti p iκb

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-p-IκB is an antibody that specifically recognizes the phosphorylated form of the inhibitor of kappa B (IκB) protein. IκB is a critical regulator of the NF-κB signaling pathway, which plays a key role in various cellular processes such as inflammation, immune response, and cell survival. The phosphorylation of IκB leads to its degradation, allowing NF-κB to translocate into the nucleus and activate target gene expression. This antibody can be used to detect the activation of the NF-κB pathway in research applications.

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5 protocols using rabbit anti p iκb

Neutrophil Protein Extraction and Analysis

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Isolated human and murine neutrophils were homogenized in protein lysis buffer (150 mM NaCl, 1% Triton X-100) (AppliChem), 0.5% Na deoxycholate (MilliporeSigma), 50 mM Tris-HCl pH 7.3 (Merck), and 2 mM EDTA (Merck) supplemented with protease (Roche) and phosphatase inhibitors (MilliporeSigma), and proteins were resolved by SDS-PAGE and then electrophoretically transferred from the gels onto PVDF membranes, which were subsequently blocked in LI-COR blocking solution and incubated with antibodies. The following antibodies were used for detection: mouse/rabbit anti-A20 (Abcam catalog ab13597 and Santa Cruz Biotechnology catalog sc-166692), rabbit anti-p52 (Cell Signaling Technology catalog 4882S), rabbit anti-pIκB (catalog 2859) and rabbit anti-IκB (catalog 4814) (Cell Signaling Technology), and mouse anti-GAPDH (Calbiochem, catalog MAB374). IRDye 680RD (catalog 926-68070) and IRDye800CW (catalog 925-32210) secondary antibodies were purchased from LI-COR. Western blots were scanned using the Odyssey CLx Imaging System and analyzed with Image Studio software (both LI-COR).

Rohwedder I., Wackerbarth L.M., Heinig K., Ballweg A., Altstätter J., Ripphahn M., Nussbaum C., Salvermoser M., Bierschenk S., Straub T., Gunzer M., Schmidt-Supprian M., Kolben T., Schulz C., Ma A., Walzog B., Heinig M, & Sperandio M. (2023). A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny. JCI Insight, 8(4), e155968.

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Quantitative Western Blot Analysis

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Proteins were extracted from 3T3-L1 adipocytes using RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich), phenylmethylsulfonyl fluoride, and Na₃VO₄. The protein concentration was determined using a bicinchoninic acid assay (Sigma-Aldrich). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking, the membrane was incubated with rabbit anti-HSP70, rabbit anti-p-IκB, rabbit anti-IκB, and mouse anti-β-actin (Cell Signaling, Danvers, MA, USA). Following this, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (Cell Signaling) and imaged using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA). The band density was normalized relative to that of β-actin.

Shin S, & Ajuwon K.M. (2023). Role of heat shock protein 70 in regulation of anti-inflammatory response to curcumin in 3T3-L1 adipocytes. Nutrition Research and Practice, 17(3), 397-407.

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Antibody Protocol for Western Blotting

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Rabbit polyclonal anti-Srgap2 antibody generated in-house by Dr. Franck Polleux was previously described (34 (link)). The following commercial antibodies were used for Western blotting: mouse anti-NFATc1 (BD Biosciences); rabbit anti-c-Src, rabbit anti-p-IκB, rabbit anti-p-ERK, rabbit anti-ERK, rabbit anti-p-JNK, rabbit anti-JNK, rabbit anti-p-p38, rabbit anti-p38, and rabbit anti-β-actin antibodies (Cell Signaling Technology); mouse anti-Myc (Millipore); anti-SLIT3 antibody (Abnova).

Shin B., Kupferman J., Schmidt E., Polleux F., Delany A.M, & Lee S.K. (2020). Rac1 inhibition via Srgap2 restrains inflammatory osteoclastogenesis and limits the clastokine, SLIT3. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 35(4), 789-800.

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Western Blot Analysis of Protein Expression

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The separated proteins were electroblotted onto nitrocellulose filter membrane and blocked for 2 h at room temperature with Tris-buffered saline containing 5% BSA. Nitrocellulose filter membrane were incubated at 4 °C overnight with antibodies, rabbit anti-KLF7 (Abcam; ab197690), rabbit anti-IL-6 (Abcam, ab214429), rabbit anti-GPR120 (Abcam; ab230869), rabbit anti-GPR40 (Thermo Fisher; PA5-75351), rabbit anti-p-IKKβ (Cell Signaling Technology; 2697S), rabbit anti-T-IKKβ (Abcam; ab124957), rabbit anti-p-IκB (Cell Signaling Technology; 2859S), rabbit anti-T-IκB (Cell Signaling Technology; 4812S), rabbit anti-p-p65 (Cell Signaling Technology; 3033S), rabbit anti-T-p65 (Cell Signaling Technology; 8242S), rat anti-GAPDH (ZSGB-BIO; TA-08), rabbit anti-TBP (Cell Signaling Technology; 44059S), rat anti-β-Actin (ZSGB-BIO; TA-10) were used at a dilution of 1:1000. The secondary antibodies (ZSGB-BIO; ZB2301 and ZB2305) were incubated at 25 °C for 2 h at a working ratio of 1:10,000.

Qiu T., Yang X., Wang J., Pan C., Chu X., Xiong J., Xie J., Chang Y., Wang C, & Zhang J. (2022). Obesity-induced elevated palmitic acid promotes inflammation and glucose metabolism disorders through GPRs/NF-κB/KLF7 pathway. Nutrition & Diabetes, 12, 23.

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Immunoblot Analysis of Inflammatory Signaling

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Immunoblot analysis was performed as described earlier [13 (link)], using the following antibodies: rabbit anti-TREM-1 (Abcam, USA), rabbit anti-MyD88 (Cell Signaling, USA), rabbit anti-TRIF (Thermo Fisher Scientific, USA), mouse anti-Syk (Abcam, USA), rabbit anti-p-Syk (Thermo Fisher Scientific, USA), rabbit anti-DAP12 (Cell Signaling, USA), rabbit anti-AKT (Cell Signaling, USA), rabbit anti-p-AKT (Cell Signaling, USA), mouse anti-TLR4 (Santa Cruz, USA), rabbit anti-TLR2 (EMD Millipore, USA), rabbit anti-p38 (Cell Signaling, USA), rabbit anti-p-p38 (Cell Signaling, USA), rabbit anti-ERK (Cell Signaling, USA), rabbit anti-p-ERK (Cell Signaling, USA), rabbit anti-IκB (Cell Signaling, USA), rabbit-anti-p-IκB (Cell Signaling, USA), mouse anti-C/EBP (Santa Cruz, USA), mouse anti-β-actin (Santa Cruz, USA), and rabbit anti-4G10 (Sigma-Aldrich, USA). Either horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody (Cell Signaling, USA) was used as a secondary antibody. R406 was purchased from Selleckchem.

Nguyen T.T., Yoon H.K., Kim Y.T., Choi Y.H., Lee W.K, & Jin M. (2020). Tryptophanyl-tRNA Synthetase 1 Signals Activate TREM-1 via TLR2 and TLR4. Biomolecules, 10(9), 1283.

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