44-280G

antibody from Invitrogen Antibodies

Targeting: FOS AP-1, c-fos

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

44-280G

Provider

Invitrogen Antibodies

Product name

Phospho-c-Fos (Thr232) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Storage

-20°C

References

Submitted references

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

Bossis G, Malnou CE, Farras R, Andermarcher E, Hipskind R, Rodriguez M, Schmidt D, Muller S, Jariel-Encontre I, Piechaczyk M
Molecular and cellular biology 2005 Aug;25(16):6964-79

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Peptide Competition. Extracts of SKBR3 cells were treated with 200 ng/mL EGF for 10 minutes and resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 3% Milk-TBST buffer for one hour at room temperature, then incubated with the c-Fos (pT232) antibody for two hours at room temperature in a 3% Milk-TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphothreonine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F (ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to c-Fos (pT232) blocks the signal, demonstrating the specificity of the antibody. While c-Fos (pT232) is phosphorylated in the basal state of SKBR3 (data not shown), EGF treatment induced an increase in the phosphorylation signal.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-c-Fos pThr232 was done on 70% confluent log phase HeLa cells treated with 20 ng of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Phospho-c-Fos pThr232 Rabbit Polyclonal Antibody (Product # 44-280G) 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 untreated cell with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry analysis of Phospho-c-Fos pThr232 showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- Phospho-c-Fos pThr232 Polyclonal Antibody (Product # 44-280G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of endogenous c-Fos (pT232) Protein at specific gene loci using Anti-c-Fos (pT232) Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-c-Fos (pT232) Rabbit Polyclonal Antibody (Product # 44-280G, 3 µg) on sheared chromatin from 2 million A431 cells treated with EGF (200 ng/mL) for 30 minutes using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoter of active IL-6, MMP9 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.