44-714

antibody from Invitrogen Antibodies

Targeting: CHUK IkBKA, IKK-alpha, IKK1, IKKA, NFKBIKA, TCF16

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

44-714

Provider

Invitrogen Antibodies

Product name

Phospho-IKK alpha (Ser176, Ser180) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

References

Submitted references

Autophagic feedback-mediated degradation of IKKα requires CHK1- and p300/CBP-dependent acetylation of p53.

Xu X, Zhang C, Xu H, Wu L, Hu M, Song L
Journal of cell science 2020 Nov 16;133(22)

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition and Induction, Immunoprecipitates prepared from lysates of Jurkat cells stimulated with 80 ng/mL TNF-alpha for 5 minutes at 37C (1-4, 6) or left unstimulated (5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C and incubated with 1:500 dilution of Phospho-IKK alpha (Ser176, Ser180) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5, 6), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphoserine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F (ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the peptide corresponding to Phospho-IKK alpha (Ser176, Ser180) blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that the phospho-signal is induced by the addition of TNF-alpha.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-IKK alpha (Ser176, Ser180) was done on 70% confluent log phase HeLa cells. The cells were treated for 20 minutes with 50 ng/mL of TNF-Alpha and fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-IKK alpha (Ser176, Ser180) Rabbit Polyclonal Antibody (Product # 44-714) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow cytometry analysis of IKK alpha [pSpS176/180] was done on Jurkat cells treated with TNF alpha (50ng/mL, 10 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with IKK alpha [pSpS176/180] Rabbit Polyclonal Antibody (44714, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

ChIP- qPCR analysis of IKK-alpha pSer176/pSer180 was performed with 10 µL of the Phospho IKK-alpha pSer176/pSer180 Rabbit polyclonal antibody (Product # 44-714) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL of TNF Alpha for one hour using the MAGnify™ Chromatin Immunoprecipitation System (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA from each ChIP sample was analyzed by StepOnePlus™ Real-Time PCR System (Product # 4376600) with primers for the promoter of active IL-8 gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.