- 701063 - Invitrogen Antibodies STAT5A antibody

Submitted references Fatty acid transport protein 2 reprograms neutrophils in cancer.

Veglia F, Tyurin VA, Blasi M, De Leo A, Kossenkov AV, Donthireddy L, To TKJ, Schug Z, Basu S, Wang F, Ricciotti E, DiRusso C, Murphy ME, Vonderheide RH, Lieberman PM, Mulligan C, Nam B, Hockstein N, Masters G, Guarino M, Lin C, Nefedova Y, Black P, Kagan VE, Gabrilovich DI
Nature 2019 May;569(7754):73-78

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Phospho-STAT5 pTyr694 in whole cell extracts of A431 cells treated with EGF (100 ng/mL, 5 min) using a Phospho-STAT5 pTyr694 recombinant rabbit monoclonal antibody (Product # 701063) at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 1). Results show a band at ~90 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Phospho-STAT5 pTyr694 in whole cell extracts of A431 cells treated with EGF (100 ng/mL, 5 min) using a Phospho-STAT5 pTyr694 recombinant rabbit monoclonal antibody (Product # 701063) at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 1). Results show a band at ~90 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of STAT5A (pY694) was done on 70% confluent log phase HeLa cells; untreated and treated (serum starved for 16 hours followed by treatment with 20 ng/mL IFN alpha for 1 hour). The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with STAT5A (pY694) Recombinant Rabbit Monoclonal Antibody (Product # 701063) at 2 µg-4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 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 Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing translocated STAT5A (pY694) in nucleus. Panel e shows competition with STAT5A (pY694) peptide. Panel f shows cytoplasmic localization of STAT5A (pY694) in untreated HeLa cells. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of STAT5A (pY694) showing staining in the cytoplasm and nucleus of paraffin-embedded human lung squamous 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 STAT5A (pY694) Monoclonal Antibody (clone 6H5L15), Recombinant (Product # 701063) diluted in 3% BSA-PBS at a dilution of 1:100 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: Immunohistochemistry analysis of STAT5A (pY694) showing staining in the cytoplasm and nucleus of paraffin-embedded mouse spleen tissue (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 STAT5A (pY694) Monoclonal Antibody (clone 6H5L15), Recombinant (Product # 701063) 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: Immunohistochemistry analysis of STAT5A (pY694) showing staining in the cytoplasm and nucleus of paraffin-embedded human diffuse large B cell lymphoma (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 STAT5A (pY694) Monoclonal Antibody (clone 6H5L15), Recombinant (Product # 701063) diluted in 3% BSA-PBS at a dilution of 1:100 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 STAT5A [pY694] was done on HeLa cells treated with IFN gamma (150 ng/ml, 15 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª STAT5A [pY694] Recombinant Rabbit Monoclonal Antibody (701063, red histogram) or with rabbit isotype control (pink histogram) at 2 µg-4 µg/million cells in 2.5% BSA. After incubation at room temperature for 2-3 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: ChIP- qPCR analysis of STAT5A (pY694) was performed with 3 µg/mL of the STAT5A (pY694) Recombinant Rabbit Monoclonal Antibody (Product # 701063) on sheared chromatin from 2 million HeLa cells treated with IFN-gamma (50 ng/mL) for 1h using the MAGnify Chromatin Immunoprecipitation System (Product # 49-2024). Normal rabbit IgG (3 µg/mL) 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 AP-1 and COX2 gene, used as positive control targets, and the inactive SAT2 satellite repeat, 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Chromatin immunoprecipitation analysis of Phospho-STAT5 (pTyr694) was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a Phospho-STAT5 alpha (pTyr694) rabbit monoclonal antibody (Product # 701063). Chromatin aliquots from ~1 x 10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify exon-1 or exon-2 of human Egr-1, or the imprinting control region (ICR) of the human H19 locus. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the human Egr-1 and H19 loci are shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag lines represent introns, and the straight line represents upstream sequence. Regions amplified by Egr-1 and H19 primers are represented by black bars. Data courtesy of the Innovators Program.

701063

Antibody data