- 44-1111G - Invitrogen Antibodies ETS1 antibody

Submitted references Phosphorylation of ETS1 by Src family kinases prevents its recognition by the COP1 tumor suppressor.

Lu G, Zhang Q, Huang Y, Song J, Tomaino R, Ehrenberger T, Lim E, Liu W, Bronson RT, Bowden M, Brock J, Krop IE, Dillon DA, Gygi SP, Mills GB, Richardson AL, Signoretti S, Yaffe MB, Kaelin WG Jr
Cancer cell 2014 Aug 11;26(2):222-34

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ETS1 (pS282/pS285) was performed by loading 20 µg of Jurkat (lane1), PC-3 (lane2) and PANC-1 (lane3) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. ETS1 (pS282/pS285) was detected at ~ 50 kDa using ETS1 (pS282/pS285) Rabbit polyclonal Antibody (Product # 44-1111G) at 1:1000 dilution in 5 % skim milk at 4ºC overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ETS1 (pS282/pS285) was performed by loading 20 µg of Jurkat (lane1), PC-3 (lane2) and PANC-1 (lane3) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. ETS1 (pS282/pS285) was detected at ~ 50 kDa using ETS1 (pS282/pS285) Rabbit polyclonal Antibody (Product # 44-1111G) at 1:1000 dilution in 5 % skim milk at 4ºC overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Lysates from Jurkat cells either untreated (1, 6), treated with hydrogen peroxide (2-5), or ionophore A23187 (7) were resolved on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 3% milk-TBST buffer for one hour at room temperature, then were incubated with the Phospho-ETS1 (Ser282/Ser285) antibody (Product # 44-1111G) overnight at 4°C in a 1% milk-TBST buffer, following prior incubation with: no peptide (1-2, 6-7), the non-phosphopeptide corresponding to the immunogen (3), a generic serine containing peptide (4), or the phosphopeptide immunogen (5). 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ETS (pS282/pS285) was done on 70% confluent log phase A549 cells. The cells were 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 ETS (pS282/pS285) Rabbit Polyclonal Antibody (Product # 44-1111G) at 1:250 dilution in 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of ETS (PS282/PS285) showing staining in the nucleus of paraffin-embedded human T cell lymphoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM 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 ETS (pS282/pS285) Rabbit Polyclonal Antibody (Product # 44-1111G) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of ETS [pS282/pS285] was done on PC-3 cells. 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 ETS [pS282/pS285] Rabbit Polyclonal Antibody (441111G, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin Immunoprecipitation (ChIP) was performed using Anti-Phospho-ETS1 pSer282/pSer285 rabbit polyclonal antibody (Product # 44-1111G, 5 µg) on sheared chromatin from 2 million Jurkat cells using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG (1 µg) 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 promoters of the active Rho-GDIbeta, MCL1 region used as positive control target gene, and the promoter region of GAPDH region, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

44-1111G

Antibody data