13-3600

antibody from Invitrogen Antibodies

Targeting: STAT5A MGF, STAT5

Provider product page for 13-3600

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Gel shift

Other assay

Antibody data

Antibody Data
Antigen structure
References [21]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [1]
Other assay [13]

Submit

Product number: 13-3600 - Provider product page
Provider: Invitrogen Antibodies
Product name: STAT5 alpha Monoclonal Antibody (ST5a-2H2)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This antibody recognizes the STAT5a protein from a variety of species including human, mouse, and rat and it reacts specifically with the STAT5a protein and does not exhibit any cross-reactivity with the highly related STAT5b protein. Cross-reactivity with other endogenous STAT isoforms has not been observed. Reactivity has been confirmed by western blotting using cell lysates derived from human A431 cells (+/- EGF treatment), NIH 3T3 cells, MCF-7 cells, and HeLa cells.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: ST5a-2H2
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

STAT5A protein structure - 13-3600 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis using STAT5 Alpha Monoclonal Antibody, Mouse (Product # 13-3600) in: Lane 1: 293T cells transfected with Stat5a. Lane 2: 293T cells transfected with STAT5b. Lane 3: untransfected 293T cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of STAT5 alpha was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR731245_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of STAT5 alpha was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa STAT5 alpha KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with a STAT5 alpha Monoclonal Antibody (ST5a-2H2) (Product # 13-3600, 1:250 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to STAT5 alpha.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of STAT5 alpha was performed by loading 20 µg of NIH/3T3 (lane1), A431 (lane2), K562 (lane3) and Ramos (lane4) cell lysate using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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. STAT5 alpha was detected at ~ 95 kDa using STAT5 alpha Mouse Monoclonal Antibody (Product # 13-3600) at 1:250 dilution in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody 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: NULL

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. Rac1 signalling switches promoter occupancy from BCL-6 to STAT5. ( A ) DLD-1 cells were transfected with Myc-Rac1-Q61L or control empty vector and 24 h later analyzed by Western blot for the subcellular distribution of STAT5 between a soluble (S) and a chromatin-bound non-soluble (NS) fraction (detection of alpha-tubulin and histone 2B served as controls). Note that active Rac1 promotes retention of STAT5 in the non-soluble chromatin fraction. ( B ) Subcellular localization of STAT5 determined by confocal fluorescence microscopy in DLD-1 cells co-transfected with DsRed-Rac1-Q61L and GFP-STAT5A. The overlay image of the DAPI, GFP and DsRed channels is shown. A microscopic field was chosen that contained side by side untransfected cells (blue nuclei), cells that transfected only with GFP-STAT5 (green cells) and cells that co-transfected with both GFP-STAT5 and DsRed-Rac1-Q61L (red cells). Note the nuclear STAT5 signal in Rac1-expressing red cells. In addition, two plots are given showing the DAPI and GFP signal intensities measured along the indicated regions of interest (ROI, white lines). The signal intensity of GFP did not increase across the nuclear DAPI region when cells expressed only GFP-STAT5 (green cells, ROI 1), whereas nuclear GFP signal clearly increased when cells co-expressed active Rac1 (red cells, ROI 2), confirming nuclear translocation of GFP-STAT5. ( C ) DLD-1 cells were co-transfected with the reporter and the indicated expression vectors, as descr

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. Regulation of expression of the CCND2, CDKN2B and SUMO1 genes. ( A ) Promoter occupancies with BCL-6 and STAT5 at the CCND2, CDKN2B and SUMO1 gene promoters was determined by ChIP with the indicated antibodies using lysates of the three indicated cell lines (see legend to Figure 2 D for details). A representative semi-qPCR of the precipitated promoter fragments and a graphical representation of the respective band intensities, * P < 0.05, are shown. A control genomic fragment from the MUTYH gene was amplified to confirm the specificity of the precipitated target gene promoters. Note that BCL-6 binds predominantly in the PAK1-lacking SW480 cells and whereas a switch to STAT5 occurs in HT29 cells with active Rac1/PAK1 signalling. ( B ) Gene expression data corresponding to the ChIP analysis of the three genes in the three cell lines. Left panel shows representative semi-quantitative reverse transcriptase-PCRs (RT-PCRs), whereas graph at the right shows the result of qPCR analysis of cDNAs collected from the three cell lines at three different splitting times. Genes encoding RNA polymerase II (pol 2) and the glycolytic enzyme phosphoglycerate kinase (PGK1) were amplified as control housekeeping genes and a pool of cDNAs mixed at equal parts from the three cell lines was used as reference for qPCR. Serial dilutions served to assure semi-quantitative conditions in the conventional RT-PCR reactions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6. Rac1 signalling controls target gene expression by inverting promoter occupancy with either BCL-6 or STAT5. The representative analysis of the SUMO1 gene is shown in the indicated three colorectal cell lines following their transfection with constructs that either activate or inhibit Rac1 signalling. Top panels show the graphical display of promoter occupancy by ChIP using either anti-BCL-6 (black columns) or STAT5 (white columns) and middle panels the respective gene expression levels (grey columns) (see legend to Figure 5 for further details), * P < 0.05. Bottom panels show Western blot analysis of the cell lysates demonstrating the expression levels of the transfected GFP, GFP-Rac1 or GFP-PAK1 constructs as well as the resulting phosphorylation status of endogenous STAT5. Note that in SW480, which lack endogenous PAK1, depletion of endogenous PAK1 by siRNAs transfection (documented in Fig. S2B ) or expression of dominant negative PAK1 has no effect on promoter-bound BCL-6, whereas re-expression of PAK1 leads to loss of BCL-6 from the SUMO1 promoter and an increase in gene expression. In the other two cell lines, inhibition of Rac1 or PAK1 are clearly correlated with more BCL-6 bound and less gene expression while activation of Rac1 or PAK1 promoted STAT5 binding to the promoters and increased transcription.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. Rac1 signalling promotes transcription by repressing BCL-6 and stimulating STAT5. ( A ) Schematic representation of the transcriptional luciferase reporter vector under the control of five consensus BCL-6 binding motifs. ( B ) DLD-1 cells were co-transfected with the reporter vector and the indicated expression vectors or siRNAs. Cells were lysed 24 h later and luciferase activity was measured and graphically displayed, * P < 0.05. The Western blot below the graph shows the levels of transfected GFP-tagged BCL-6, Rac1-L61, PAK1 kinase dead (kd) or PAK1 constitutively active (ca) mutants. Detection of endogenous alpha-tubulin served as loading control. The small insert beside the graph shows a Western blot of endogenous BCL-6 to document the efficiency of its siRNA-mediated depletion.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3. Correlation of Rac1 signalling and activation of PAK1, BCL-6 or STAT5 in different cell lines. Equivalent lysate quantities of DLD-1, SW480 and HT29 colorectal cells were separated by gel electrophoresis and analysed by Western blot using the indicated antibodies to compare protein levels. The active Rac1 fraction was obtained by CRIB-pull down assays, as described ( 6 ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Binding activity of two potential STAT5 binding sites located within the CSN1S1 distal upstream region. (A) Representative EMSA. Mammary nuclear extract (22ug) from rabbits in early lactation were incubated with the indicated labelled 32 P probes in the presence or absence of STAT5a or STAT5b antibodies (1 or 0.5 ug, respectively). Complexes were analysed on non denaturating 5% acrylamide gels in 0.25xTBE. The lower panel is an overexposed image of the upper one to show the weak interaction between D1 and nuclear extracts and the supershifts with STAT5 antibodies. (B) Competition assays with increasing amounts of unlabelled C, D2, D1 and D1m using 32 P-C or 32 P-D2, are indicated in each graph. Results are expressed as a percentage of the radioactivity bound in the absence of unlabelled oligonucleotides and plotted as a function of the log (ng oligonucleotide) in the reaction.