71-6900

antibody from Invitrogen Antibodies

Targeting: STAT5A MGF, STAT5

Provider product page for 71-6900

Western blot

ELISA

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [39]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Other assay [30]

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Product number: 71-6900 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-STAT5 alpha (Tyr694) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat, Bovine
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 0.25 mg/mL
Storage: -20°C

STAT5A protein structure - 71-6900 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 7. DN-Pak1-TG mammary glands show decreased Stat5 tyrosine phosphorylation and expression. (A and B) Immunohistochemical staining of phospho-Stat5a in mammary glands obtained from wild-type and Pak1-TG mice on lactation day 5. (C) Effect of Pak1 inactivation on the levels of Stat5a and phosphorylated Stat5a by immunoblot analysis. Bar: (A and B) 20 mum.

Supportive validation

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Experimental details: Fig. 2 Combination of hepatic GH resistance and hypercortisolism causes peripheral lipodystrophy in DKO animals. (A) Histology of WAT. Epigonadal WAT of 12-month-old mice was stained with hematoxylin and eosin. (B) Quantification of WAT cell density. Hematoxylin and eosin-stained sections were used to analyze cell density using HistoQuest image analysis (TissueGnostics GmbH, Vienna, Austria). (C) Levels of FFA were determined in 2-month-old mice using colorimetric assays. (D) Levels of GH and IGF-1 were determined by ELISA. Levels of corticosterone (Cort) and ACTH were determined by radioimmunoassay (n = 8/genotype/time point). (E) Representative western blotting analysis of WAT homogenates from 2-month-old mice. Expression levels of STAT5 and GR proteins were determined using specific antibodies. HSC-70 served as the loading control. (F) Relative mRNA levels of Hsl , Atgl , and Plin were quantified by qRT-PCR in WAT from 6-month-old mice. Ct values were normalized to GAPDH (DeltaCt method; n = 4/genotype). * P < 0.05; ** P < 0.01; *** P < 0.001.

Supportive validation

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Experimental details: Figure 2 p-STAT5 Tyr694 and p-STAT3 Tyr705 expression upon GH treatment compared to control mice. A Western Blot of p-STAT5 Tyr694 expression in Stat5ab +/+ (n = 2), Stat5ab +/Delta (n = 2) and Stat5ab Delta/Delta (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. B IHC of p-STAT5 Tyr694 in sections of the livers (n = 3) used in A. Red, AEC; blue, hematoxylin. Size bar: 100 um. High magnifications (60x) are shown in insets. C Quantification of p-STAT5 Tyr694 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software ( Stat5ab +/+ , control; number of cells n = 3276, GH; number of cells n = 2286; Stat5ab +/Delta control; number of cells n = 2577, GH; number of cells n = 3549; Stat5ab Delta/Delta control; number of cells n = 3299, GH; number of cells n = 2200). The highest arbitrary expression values from A and B were set to 100 for direct comparison. D Western blot of p-STAT3 Tyr705 expression in Stat5ab +/+ (n = 2), Stat5ab +/Delta (n = 2) and Stat5ab Delta/Delta (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. E IHC of p-STAT3 Tyr705 in sections of the livers (n = 3) used in D. Red, AEC; blue, hematoxylin. Size bar: 100 um. High magnifications are shown in insets (60x). F Quantification of p-STAT3 Tyr705 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software ( Stat5ab +/+ , control; number of cells n = 2634, GH; n

Supportive validation

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Experimental details: Figure 3 ( A ) Cytospin preparations of HEL cells and SET-2 cells were stained overnight with an anti-pSTAT5 antibody using immunocytochemistry. Scale bar: 20 uM. ( B ) HEL cells and SET-2 cells were stained with an anti-pSTAT5 Alexa-647 antibody for 30 min at room temperature and intracellular expression levels were analyzed by flow cytometry. ( C ) Nuclear (NE) and cytoplasmic (CE) fractions of HEL or SET-2 cells were analyzed for expression of phosphorylated STAT5 (pSTAT5), total STAT5a and total STAT5b by Western blotting. Antibodies against RAF-1 (cytoplasm) and TOPO-1 (nucleus) were used as fraction controls. The columns show the densitometry for pSTAT5. Uncropped blots are shown in Figure S1 .

Supportive validation

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Experimental details: Figure 4 Ba/F3 cells modified to express JAK2 V617F or CALRdel52 were incubated with various JAK2 or STAT5 inhibitors (as indicated) for four hours at 37 degC. Thereafter, the expression of activated STAT5 (pSTAT5), total STAT5 (STAT5) and heat shock cognate 70 (HSC70), which was used as loading control, was analyzed using Western blotting. The results were quantified using densitometry and are expressed as the ratio of activated and total STAT5 normalized to the results obtained with control cells (Ctrl). Uncropped blots are shown in Figure S2 .

Supportive validation

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Experimental details: Figure A3 Wild type (wt) Ba/F3 cells or Ba/F3 cells transfected with either JAK2 V617F or CALRdel52 were incubated overnight in medium without IL-3. After, cell lysates were subjected to Western blotting using antibodies against pSTAT5, STAT5 and HSC70. The columns show the levels of pSTAT5 in percent of HSC70 as analyzed by densitometry. Uncropped blots are shown in Figure S3 .

Supportive validation

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Experimental details: Figure A5 Ba/F3 cells were kept in medium without IL-3 overnight and split in the morning. One half was incubated with 10 ng/mL IL-3 for 40 min (Ba/F3 + IL-3), while the other half was kept in medium without IL-3 (Ba/F3 - IL-3). Thereafter, cells were analyzed for expression of pSTAT5 either by flow cytometry ( A ) or Western blotting ( C ). In ( A ), an Alexa-647-labeled anti-pSTAT5 antibody was used (with IL-3: blue histogram; without IL-3: open blue histogram). mIgG1 Alexa-647 was used as an isotype control (open black histogram) in Ba/F3 + IL-3 cells ( A ). Human lymphoma cell lines with detectable pSTAT5 levels (KOPT-K1 and MYLA) or no detectable pSTAT5 (HUT78 and HH) were also analyzed for pSTAT5 expression using flow cytometry ( B ) and Western blotting ( D ). In Western blot experiments, HSC70 served as a loading control. The columns show the levels of pSTAT5 in percent of HSC70 as analyzed by densitometry. Uncropped blots are shown in Figure S4 .

Supportive validation

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Experimental details: Figure 2 Collagen ligand density does not modulate PRL signals to ERK1/2 or FAK A-C. T47D were cells plated on 25 kPa polyacrylamide gels coated with either 50, 200, or 800 mug/ml collagen-I, serum starved for 24h, then treated +- PRL (4 nM) for 15 min. Cell lysates were immunoblotted with the indicated antibodies. Top panels: Representative immunoblots. Bottom panels: Quantification of immunoblots by densitometry. Means +- S.E.M. n = 3. Different letters represent significant differences between treatments, p

Supportive validation

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Experimental details: Figure 5 Stat5 phosphorylation is reduced in Miz1 Delta POZ mammary glands and in shMiz1 HC11 cells. ( A ) Analysis of Stat5a/b mRNA expression in Miz1 Delta POZ (DeltaPOZ) and control mammary gland tissue ( Ctr ). ( B ) Immunoblot analysis of Stat5 in wildtype and Miz1 Delta POZ mammary glands. ( C ) Immunohistochemical staining of pStat5 in the mammary gland tissue from wildtype and Miz1 Delta POZ animals. ( D ) Western blots from HC11 cells stably transfected with a scrambled short hairpin (sh) RNA (shscr) or a Miz1 shRNA (see also Fig. 4 E and F ). Numbers indicated are fold changes of band intensities obtained by densitometry (see Materials and Methods). Quantitative RT-PCR for the prolactin receptor ( E ; Prlr ), the Supressors of cytokine signalling ( Socs ) 1, 2 and 3 and caveolin-1 ( F ; Cav1 ), and for ErbB4 ( G ). Lactation day 6 samples were used in all in vivo experiments. Scale bar in C: 50 um.

Supportive validation

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Experimental details: Fig 1 pSTAT5 binds promoters and enhancers of key erythroid genes in concert with GATA1 and KLF1. (A) 302 peaks were annotated with respect to the nearest TSS as defined by RefSeq. Only ~23% of peaks reside within gene promoters (1 kb upstream or 100 nt downstream of a TSS). The majority are intronic or distal (within 50 kb of a gene body) consistent with enhancer occupancy. (B) De novo motif discovery on all 302 peaks using MEME (see Methods ) identified highly significant enrichment of a (i) palindromic GAS element (TTCYMRGAA), a (ii) GATA binding site (WGATAR), and a (iii) KLF binding site (CCMCRCCCN). No other significantly enriched motifs were found. (C) Venn diagram and of erythroid co-occupancy between pSTAT5, KLF1 and GATA1 at key erythroid enhancers and promoters. KLF1 ChIP-seq was generated in K1-ER cells and GATA1 ChIP-seq was generated in G1-ER4 cells after induction with tamoxifen. Co-occupancy defined as ChIP summits within 500 bp. (D) Density heat-map of ChIP signal centred on pSTAT5 peak summits from GATA1 and KLF1 as above (C) and additional STAT5 ChIP in other murine tissues. The Y-axis represents individual peak regions, and the X-axis represents 10 kb surrounding the summit. Read intensities were normalized to number of reads in dataset and hierarchically clustered according to intensity within 500 bp of peak centre.

Supportive validation

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Experimental details: Fig 3 Immediate direct transcriptional targets of EPOR-pSTAT5 signalling. (A) ChIP-seq and 4sU-RNA-seq in erythroid cells following 30 mins of EPO induction across the Cish gene. Read density per million mapped reads (y-axis) for pSTAT5 (ochre), KLF1 (orange) and GATA1 (brown) ChIP. Coloured bars above each track represent peak summits as called by MACS2. Read density profiles (y-axis) for 4sU-RNA-seq from J2E cells (blue) and J2E cells post EPO-induction (red) are displayed for both forward (+ values) and reverse (- values) strands. A schematic of gene structure and alternative transcripts from RefSeq is shown in black with scale bar. The green bar represents the region selected for CAGE tag counts shown in (C). (B) qRT-PCR for Cish primary pre-spliced transcripts and processed mRNA over a 24-hour time course following EPO stimulation. (C) CAGE tag counts expressed as relative log expression (RLE) over the indicated pSTAT5-occupied promoter (green bar in panel A) from 0 to 24 hours post EPO stimulation. ChIP-seq and 4sU-RNA-seq across the Bcl2l1 (D) and Suv420h2 (G) genes. Overall design and colour coding of tracks is the same as for (A). Purple bars indicate pSTAT5-occupied intronic enhancers. Dynamic EPO-induced CAGE tags over this region are shown in S4 Fig . qRT-PCR for Bcl2l1 (E) and Suv420h2 (H) primary pre-spliced transcripts and processed mRNA over a 24-hour time course following EPO stimulation as in (B). CAGE tag counts for Bcl2l1 (E) and Suv420h2

Supportive validation

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Experimental details: Fig 4 Delayed pSTAT5-dependent and independent EPO-induced gene expression. ChIP-seq and 4sU-RNA-seq in erythroid cells following 30 mins of EPO induction across the Gypc (A) and Thbs1 (D) genes. The overall design and colour coding of tracks is the same as for Fig 3 . qRT-PCR and CAGE tag counts as in Fig 3B and 3C for Gypc and Fig 3E and 3F for Thbs1 respectively. Bidirectional CAGE tags over the intronic enhancer (purple bar in panel A) are shown in S4C Fig . Gypc provides an example of delayed transcriptional activation by pSTAT5, whereas Thbs1 appears to be activated independently of pSTAT5.