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α iκbα

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α-IκBα is a primary antibody that specifically recognizes the alpha isoform of IκBα (Inhibitor of NF-κB alpha). IκBα is a regulatory protein that binds to and masks the nuclear localization signals of NF-κB, thereby keeping NF-κB inactive in the cytoplasm.

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Lab products found in correlation

α gapdh, by Cell Signaling Technology (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions) Mln4924, by Active Motif (1 mentions) α ve cadherin xp, by Cell Signaling Technology (1 mentions) α p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions) α rhoa, by Cell Signaling Technology (1 mentions) α rhoc, by Cell Signaling Technology (1 mentions) α rhob, by Santa Cruz Biotechnology (1 mentions) Bay 11 7085, by Cayman Chemical (1 mentions) Acti stain 670 phalloidin, by Cytoskeleton (1 mentions) Csn5i 3, by Novartis (1 mentions) α p p65, by Cell Signaling Technology (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Y 27632, by Merck Group (1 mentions) α flag m2, by Merck Group (1 mentions) α γh2ax, by Cell Signaling Technology (1 mentions) 7 aad, by BioLegend (1 mentions) Goat α mouse hrp, by Agilent Technologies (1 mentions) α rabbit alexafluor 488, by Thermo Fisher Scientific (1 mentions)

4 protocols using α iκbα

1

Measuring IκBα Degradation in HEK 293 Cells

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HEK 293 cells were maintained in DMEM supplemented with 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine. Confluent cells were split at 1:12 into 6-well plates and used for experiments on day 3. Untreated and oxidized ligands were added to cells to final concentrations of 200 ng/mL and 20 ng/mL for LTα and TNF respectively, and incubated for 20 minutes at 37 °C. Cells were then washed once and gently sheared from the plate with ice-cold PBS, then centrifuged and resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer. Cell lysates were normalized to equal protein concentrations, and 60 µg were loaded onto 4–12% Bis-Tris gradient gels for Western blot analysis. IκBα was detected using horseradish peroxidase (HRP) conjugated rabbit α-IκBα (Cell Signaling Technology, #9242, 1:1000) and α-Rabbit IgG (Amersham, 1:10,000). Full gels are shown in Supplementary Fig. 16.
Lewis A.K., Dunleavy K., Senkow T.L., Her C., Horn B.T., Jersett M.A., Mahling R., McCarthy M.R., Perell G.T., Valley C.C., Karim C.B., Gao J., Pomerantz W.C., Thomas D.D., Cembran A., Hinderliter A, & Sachs J.N. (2016). Oxidation increases the strength of the methionine-aromatic interaction. Nature chemical biology, 12(10), 860-866.
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2

Investigating Cell-Cell Adhesion Regulators

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The following antibodies were used in this study: αVE-cadherin XP (#2500), αRhoA (#2117), αRhoC (#3430), αCullin-3 (#2759), αp44/42 MAPK (ERK1/2) (#9102), αpMLC2 Ser19 (#3671), αGAPDH (#2118), αIκBα (#4841) and αpp65 (#93H1) (all from Cell Signaling Technology); αRhoB (#sc-8048 and #sc-180), αCullin-1 (#sc-17775), αICAM (#sc-8439) (all from Santa Cruz Biotechnology); αCullin-2 (#610778) and αp38 (#61268) (BD Transduction Laboratories) and αpp38 (#09–272) (Millipore).
Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies (Dako) were used as secondary antibodies for western blotting. For immunofluorescent staining DAPI (Thermo Fisher scientific), Alexa 488-secondary antibody (anti-rabbit and anti-mouse, Invitrogen) and Acti-stain 670 phalloidin (Cytoskeleton) were used.
The following inhibitors and cytokines were used in this study: CSN5i-3 (Novartis), MLN4924 (Active Biochem), Y27632 (#Y0503) (Sigma), TNF-α (#300-01 A) (PeproTech), BAY11-7085 (Cayman Chemical).
Majolée J., Pronk M.C., Jim K.K., van Bezu J.S., van der Sar A.M., Hordijk P.L, & Kovačević I. (2019). CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity. Scientific Reports, 9, 8131.
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3

Apoptosis Induction Assay Protocol

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Chemicals and cytokines used were staurosporine, cycloheximide, doxorubicin, DAPI, H2O2, Nutlin-3, polybrene, propidium-iodide, doxocycline, puromycin (all Sigma), AnnexinV–APC, AnnexinV-488, 7-AAD (Biolegend), etoposide, camptothecin (Alexis). Antibodies: α-IκBα (#9242), α-PARP1 (#9532), α-GAPDH (#2118), α-Histone H3 (Ser10) (#9701), α-γH2A.X (#2577), pChk1 Ser345 (#2341), Chk1 (#2345) (Cell Signaling); α-p53 (sc-6243), α-tubulin (sc-32293) (Santa Cruz Biotechnology); α-PDCD5 (Proteintech); α-LaminB1 (gift from Peter Gruber); α-Flag (M2) (Sigma), α-p21 (#554262) (BD Pharmingen); α-rabbit AlexaFluor 488 (Invitrogen), goat α-rabbit HRP, goat α-mouse HRP (Dako). SiRNA targeting human PDCD5 (5′-CUAAAGCAGUAGAGAAUUATT-3′) was synthesised by Microsynth and transfected into 293T cells using Metafectene (Biontex) according to the manufacturer′s instructions.
Bock F.J., Tanzer M.C., Haschka M.D., Krumschnabel G., Sohm B., Goetsch K., Kofler R, & Villunger A. (2015). The p53 binding protein PDCD5 is not rate-limiting in DNA damage induced cell death. Scientific Reports, 5, 11268.
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4

Measuring IκBα Degradation in HEK 293 Cells

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HEK 293 cells were maintained in DMEM supplemented with 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine. Confluent cells were split at 1:12 into 6-well plates and used for experiments on day 3. Untreated and oxidized ligands were added to cells to final concentrations of 200 ng/mL and 20 ng/mL for LTα and TNF respectively, and incubated for 20 minutes at 37 °C. Cells were then washed once and gently sheared from the plate with ice-cold PBS, then centrifuged and resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer. Cell lysates were normalized to equal protein concentrations, and 60 µg were loaded onto 4–12% Bis-Tris gradient gels for Western blot analysis. IκBα was detected using horseradish peroxidase (HRP) conjugated rabbit α-IκBα (Cell Signaling Technology, #9242, 1:1000) and α-Rabbit IgG (Amersham, 1:10,000). Full gels are shown in Supplementary Fig. 16.
Lewis A.K., Dunleavy K., Senkow T.L., Her C., Horn B.T., Jersett M.A., Mahling R., McCarthy M.R., Perell G.T., Valley C.C., Karim C.B., Gao J., Pomerantz W.C., Thomas D.D., Cembran A., Hinderliter A, & Sachs J.N. (2016). Oxidation increases the strength of the methionine-aromatic interaction. Nature chemical biology, 12(10), 860-866.
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