44-390G

Bao YN, Cao X, Luo DH, Sun R, Peng LX, Wang L, Yan YP, Zheng LS, Xie P, Cao Y, Liang YY, Zheng FJ, Huang BJ, Xiang YQ, Lv X, Chen QY, Chen MY, Huang PY, Guo L, Mai HQ, Guo X, Zeng YX, Qian CN
Cell cycle (Georgetown, Tex.) 2014;13(12):1958-69

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia.

Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M, Bunin NJ, Bunda S, Finklestein JZ, Gorr TA, Mehta P, Schmid I, Kropshofer G, Corbacioglu S, Lang PJ, Klein C, Schlegel PG, Heinzmann A, Schneider M, Starý J, van den Heuvel-Eibrink MM, Hasle H, Locatelli F, Sakai D, Archambeault S, Chen L, Russell RC, Sybingco SS, Ohh M, Braun BS, Flotho C, Loh ML
Nature genetics 2010 Sep;42(9):794-800

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Antibody-Peptide Competition. Extracts of 3T3-L1 cells untreated (1) or treated with 20 ng/mL LIF for 10 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with the STAT5 [pY694] antibody (Product # 44-390G) for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). 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 STAT5 [pY694] blocks the signal, verifying the specificity of the antibody. The data also show the up-regulation of the signal by treatment with LIF in this cell system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-STAT5 pTyr694 was done on 70% confluent log phase HeLa cells treated with 5uM of TNF-alpha for 30 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-STAT5 pTyr694 Rabbit Polyclonal antibody (Product # 44-390G) 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 cytoplasmic and nuclear localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of STAT5 (pY694) showing staining in the 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 STAT5 (pY694) polyclonal antibody (Product # 44-390G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC 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 STAT5 [pY694] was done on HeLa cells treated with IFN gamma (100ng/mL, 30 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 STAT5 [pY694] Rabbit Polyclonal Antibody (44390G, red histogram) or with rabbit isotype control (yellow 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)
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Experimental details: ChIP- qPCR analysis of STAT5 (pTyr694) was performed with 10 µL of the STAT1 (pTyr694) Rabbit polyclonal antibody (Product # 44-390G) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL IFN Gamma treated 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 AP1 gene, used as positive control target, and the inactive 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: ChIP- qPCR analysis of STAT5 pTyr694 was performed with 10 µL of the Phospho-STAT5 pTyr694 Rabbit polyclonal antibody (Product # 44-390G) on sheared chromatin from 2 million HeLa cells treated with 100 ng/mL of IFN gamma for 45 minutes 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 COX1, AP1 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL