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Stat5a

Manufactured by Santa Cruz Biotechnology
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STAT5A is a transcription factor that plays a key role in the regulation of gene expression. It is involved in the signaling pathways that mediate the effects of various cytokines and growth factors. STAT5A is crucial for cellular processes such as cell growth, differentiation, and survival.

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Lab products found in correlation

Stat5b, by Santa Cruz Biotechnology (5 mentions) P stat5, by Cell Signaling Technology (2 mentions) Phenol red free rpmi 1640 media, by Thermo Fisher Scientific (2 mentions) β actin antibody, by Santa Cruz Biotechnology (2 mentions) Rpmi 1640 glutamax, by Thermo Fisher Scientific (2 mentions) Prlr antibody, by Merck Group (2 mentions) Nitrocellulose membrane, by Merck Group (1 mentions) C myc antibody, by Santa Cruz Biotechnology (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) α tubulin, by Santa Cruz Biotechnology (1 mentions) Hdac1, by Merck Group (1 mentions) Histone h3, by Abcam (1 mentions) Hdac3, by Cell Signaling Technology (1 mentions) Acetylated histone h3 ac h3, by Merck Group (1 mentions) Acetylated histone h4 ac h4, by Merck Group (1 mentions) Hdac2, by Thermo Fisher Scientific (1 mentions) Anti mouse igg peroxidase, by Merck Group (1 mentions) Rna polymerase 2, by Santa Cruz Biotechnology (1 mentions) Anti rabbit igg peroxidase, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-STAT5A (8 citations) Rabbit anti-STAT5A (2 citations)

17 protocols using stat5a

1

Western Blot Analysis of Protein Expression

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The cells were lysed in RIPA buffer. The protein concentration was determined by the Bradford method with BSA (Sigma) as the standard. Equal amounts of cell extract were subjected to electrophoresis in SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Merck Millipore). The membrane was blocked and then incubated with GAPDH (Ambion, Thermo Fisher Scientific Inc.), phospho-STAT5 (Tyr694) (pY-STAT5) (Cell Signaling Technology, Danvers, MA), STAT5A, and c-Myc antibodies (Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. This was followed by incubation for 1 hr with appropriate secondary antibodies. Antibody binding was detected with an enhanced chemiluminescence kit (Pierce, Thermo Fisher Scientific Inc., Waltham, MA).
Chen J.J., Zhou W., Cai N, & Chang G. (2017). In Vivo Murine Model of Leukemia Cell-Induced Spinal Bone Destruction. BioMed Research International, 2017, 3521481.
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2

Antibody Validation for Western Blot and ChIP

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Antibodies used for western blot and ChIP were pSTAT5 (Cell Signaling Technology 9351), STAT5A (Santa Cruz Biotechnology sc-1081), STAT5B (Santa Cruz Biotechnology sc-1656), STAT5A+B (Santa Cruz Biotechnology sc-835), RNA polymerase II (Santa Cruz Biotechnology sc-899 and sc-900), TBP (Santa Cruz Biotechnology sc-273), histone H3 (Abcam ab1791), acetylated histone H3 (Ac-H3; Millipore 06–599), acetylated histone H4 (Ac-H4; Millipore 06–866), α-tubulin (Santa Cruz Biotechnology sc-32293), HDAC1 (Millipore 05–100), HDAC2 (Invitrogen 51–5100), HDAC3 (Cell Signaling Technology 2632), FLAG (M2, SIGMA F-1804), Brd2 (Bethyl A302–583A) and IgG from rabbit serum (SIGMA I-5006; isotype control for ChIP). Secondary antibodies for western blot were anti-Rabbit IgG-Peroxidase (SIGMA A-0545) and anti-Mouse IgG-Peroxidase (SIGMA A-8924).
Pinz S., Unser S., Buob D., Fischer P., Jobst B, & Rascle A. (2015). Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function. Nucleic Acids Research, 43(7), 3524-3545.
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3

STAT5A Chromatin Immunoprecipitation in HIV-1 Infected Cells

Cited 1 time
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Latently infected cultured TCM were left untreated or incubated for 3 hr with IL-2 ± 100 μM HODHBt. After 15 min, 1% paraformaldehyde crosslinking in media and sonication, ChIP was performed against STAT5A (Santa Cruz Biotechnology, sc-1081). Paired-end reads were aligned to hg19 using Bowtie 2 (Langmead and Salzberg, 2012 (link)) and compiled using MACS2 (Zhang et al., 2008 (link)). Reads which failed to map to hg19 were mapped to HIV-1 NL4-3 using Bowtie 1 (Langmead et al., 2009 (link)). See Supplemental Experimental Procedures for additional details.
Bosque A., Nilson K.A., Macedo A.B., Spivak A.M., Archin N.M., Van Wagoner R.M., Martins L.J., Novis C.L., Szaniawski M.A., Ireland C.M., Margolis D.M., Price D.H, & Planelles V. (2017). Benzotriazoles Reactivate Latent HIV-1 through Inactivation of STAT5 SUMOylation. Cell reports, 18(5), 1324-1334.
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4

Exploring Receptor Signaling Pathways

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RPMI 1640-GlutaMAX and Phenol-red free RPMI 1640 media were obtained from ThermoFisher Scientific. Charcoal stripped fetal bovine serum was purchased from Atlanta Biologicals. ERα, STAT5a, STAT5b, STAT5, β-actin antibodies and inhibitors of PI3K (Wortmannin) and JAK2 (AG-490) and c-SRC (PP1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho (p)-ERα (Ser118 and Ser167), pSTAT5, pERK1/2, pMEK1/2, ERK1/2, pAKT, pEGFR (Y1068), pEGFR (Y845) and p-Tyrosine antibodies and U1026 (MEK1/2 inhibitor) were purchased from Cell Signaling. ICI 182,780 (Fulvestrant) and PRLR antibody were obtained from Sigma-Aldrich. Human PRL antibody were obtained from National Hormone and Peptide Program, Harbor-UCLA Med. Ctr., Torrance, CA 90502.
Kavarthapu R, & Dufau M.L. (2016). Role of EGF/ERBB1 in the transcriptional regulation of the prolactin receptor independent of estrogen and prolactin in breast cancer cells. Oncotarget, 7(40), 65602-65613.
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5

STAT5A Chromatin Immunoprecipitation in HIV-1 Infected Cells

Cited 1 time
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Latently infected cultured TCM were left untreated or incubated for 3 hr with IL-2 ± 100 μM HODHBt. After 15 min, 1% paraformaldehyde crosslinking in media and sonication, ChIP was performed against STAT5A (Santa Cruz Biotechnology, sc-1081). Paired-end reads were aligned to hg19 using Bowtie 2 (Langmead and Salzberg, 2012 (link)) and compiled using MACS2 (Zhang et al., 2008 (link)). Reads which failed to map to hg19 were mapped to HIV-1 NL4-3 using Bowtie 1 (Langmead et al., 2009 (link)). See Supplemental Experimental Procedures for additional details.
Bosque A., Nilson K.A., Macedo A.B., Spivak A.M., Archin N.M., Van Wagoner R.M., Martins L.J., Novis C.L., Szaniawski M.A., Ireland C.M., Margolis D.M., Price D.H, & Planelles V. (2017). Benzotriazoles Reactivate Latent HIV-1 through Inactivation of STAT5 SUMOylation. Cell reports, 18(5), 1324-1334.
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6

Immunoblotting Adiponectin Signaling Pathway

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Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma-Aldrich (St. Louis, MO). Bovine and fetal bovine sera were purchased from HyClone (Thermo Scientific, Logan, UT). For immunoblotting, STAT5A, MAPK/ERK, and PPARγ antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-adiponectin antibody was obtained from Thermo Scientific (Rockford, IL). Anti-phospho-Akt-Ser473(AktpS(473)), total Akt, and monocyte chemotactic protein-1 (MCP-1) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). With the exception of the mouse monoclonal anti-PPARγ IgG, all other antibodies were rabbit polyclonal IgGs, and peroxidase-conjugated secondary antibodies to both species were purchased from Jackson ImmunoResearch (West Grove, PA). The BCA and enhanced chemiluminescence kits were from Thermo Scientific.
Richard A.J., Burris T.P., Sanchez-Infantes D., Wang Y., Ribnicky D.M, & Stephens J.M. (2014). Artemisia extracts activate PPARγ, promote adipogenesis, and enhance insulin sensitivity in adipose tissue of obese mice. Nutrition (Burbank, Los Angeles County, Calif.), 30(0 0), S31-S36.
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7

Comprehensive Immunohistochemical Analysis of Mammary Tissue

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Samples were collected and fixed overnight in 10% neutral buffered formalin and processed into 5 micron sections. Mammary structures were assessed in hematoxylin and eosin (H&E) stained tissue sections, and protein expression was visualized by immunohistochemistry as previously described (Arendt et al., 2011 (link)) using antibodies against the following antigens: Cyclin D1, CP236B, Biocare Medical, (1:200); ER, #SC-542, Santa Cruz Biotechnology (1:500); PR, A0098, Dako (1:500); pAKTS473, #3787, Cell Signaling Technologies (1:50); pERK1/2, #9101, Cell Signaling Technologies (1:400); STAT5A, #SC-1081, Santa Cruz Biotechnology (1:5000). For CD8 and F4/80, frozen sections were stained. Tissues were embedded in OCT and frozen in a dry ice/methanol bath. Seven micron sections were cut, fixed with acetone at −20°C for 20 min, blocked with 5% normal goat serum and incubated overnight with either anti-CD8, Clone 53–6.7, Invitrogen (1:1000) or anti-F4/80, Clone BM8, Biolegend, (1:250). Slides were then processed in the same manner as for the paraffin sections.
Campbell K.M., O’Leary K.A., Rugowski D.E., Mulligan W.A., Barnell E.K., Skidmore Z.L., Krysiak K., Griffith M., Schuler L.A, & Griffith O.L. (2019). A Spontaneous Aggressive ERα+ Mammary Tumor Model Is Driven by Kras Activation. Cell reports, 28(6), 1526-1537.e4.
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8

ChIP-Seq Analysis of STAT Binding

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ChIP assays were carried out using the ChIP-IT Express Enzymatic kit (Active Motif) using a dounce homoginizer to lyse cells. Optimal enzymatic digestion of chromatin from MDA-MB-231 cells was empirically determined to occur after 10 min, yielding sheared chromatin that migrated between 200 and 1500 bp on an agarose gel. Equal DNA concentrations corresponding to 1.5 μg were applied to each set of immunoprecipitation reactions, which included either normal rabbit IgG, STAT3, or STAT5A antibody (sc-2027, sc-7179X, or sc-1081X, respectively; Santa Cruz Biotechnology). Samples were incubated with magnetic beads overnight at 4°C with end-over-end rotation. After reversal of cross-links, DNA precipitation, and clean-up, enriched DNA and input were analyzed by quantitative real time PCR with primers spanning the predicted GAS site, as well as primers specific to a region of the LKB1 promoter that does not contain a putative STAT binding motif (Table 
2). The efficiency of each primer set was tested by producing a standard curve from two-fold dilutions of input, and the integrity of products was verified by agarose gel electrophoresis. Fold enrichment relative to IgG was calculated for immunoprecipitated samples, and data are presented normalized to values obtained for the negative binding region.
Linher-Melville K, & Singh G. (2014). The transcriptional responsiveness of LKB1 to STAT-mediated signaling is differentially modulated by prolactin in human breast cancer cells. BMC Cancer, 14, 415.
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9

ChIP-seq and qPCR Analysis of Mammary Tissue

Cited 2 times
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Frozen-stored mammary tissues collected at lactation day 1 (L1) were ground into powder. Chromatin was fixed with formaldehyde (1% final concentration) for 15 min at room temperature, and then quenched with glycine (0.125 M final concentration). Samples were processed as previously described (35 (link)). The following antibodies were used for ChIP-seq: CTCF (Millipore, 07-729 and Abcam, ab70303), SMC1 (Bethyl, A300-055A), STAT5A (Santa Cruz Biotechnology, sc-1081), GR (Thermo Scientific, PA1-511A), H3K27ac (Abcam, ab4729) and RNA polymerase II (Abcam, ab5408). Libraries for next-generation sequencing were prepared and sequenced with a HiSeq 2500 instrument (Illumina) (35 (link)). ChIP-seq data analysis was done as described (36 (link)), except for CTCF samples, where the parameters -m 3 and –best were used for Bowtie aligner (37 (link)) (version 1.1.2).
Immunoprecipitated DNA was used to ChIP-Real-time quantitative PCR (qRT-PCR) and analyzed by real-time PCR with SYBR green supermix (Biorad). The PCR product was amplified using the following specific primer pairs for the promoter of Sult1d1, forward, 5΄-TTAATGGAAGCCTCGTTCTCTG-3΄ and reverse 5΄-AACATCCACAGCACCTTCTC-3΄.
Lee H.K., Willi M., Wang C., Yang C.M., Smith H.E., Liu C, & Hennighausen L. (2017). Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice. Nucleic Acids Research, 45(8), 4606-4618.
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10

Protein Expression Analysis in MV4-11 Cells

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Total protein extracts from MV4-11 cell lines were harvested using RIPA lysis buffer with protease inhibitors, and protein concentration was determined using a Bio-Rad Protein Assay Dye Reagent kit. SDS-denatured protein was separated via gel electrophoresis and transferred onto a nitrocellulose membrane. Protein was detected via overnight antibody staining with the following antibodies: STAT5A (Santa Cruz L-20), Drosha (Cell Signaling D28B1), Ago2 (Cell Signaling C34C6), p53 (Santa Cruz FL-393), and Actin (Sigma A5441).
Wallace J., Hu R., Mosbruger T.L., Dahlem T.J., Stephens W.Z., Rao D.S., Round J.L, & O’Connell R.M. (2016). Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth. PLoS ONE, 11(4), e0153689.
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