P stat5

Manufactured by Cell Signaling Technology

Sourced in United States

P-STAT5 is a phosphorylation-specific antibody that detects the phosphorylated form of STAT5 protein. STAT5 is a transcription factor that plays a critical role in cell signaling pathways.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

P-STAT5 (Tyr694) (11 citations) P-STAT5 antibodies (2 citations) P-STAT5 (Y694) (13 citations) P-Y694/699-STAT5 (2 citations) Anti-p-STAT5 (20 citations) STAT5 (129 citations)

116 protocols using p stat5

Signaling Dynamics in FDH Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

FDH cells expressing βc and WT or P248L IL3Rα were stimulated with 100 ng/mL IL3 for specified time points up to 60 minutes before lysis with NP-40 lysis buffer [1% NP-40 (v/v), 150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 8.0, and 10% glycerol (v/v)] for 20 minutes on ice. Lysates were cleared by centrifugation and immunoblotted with antibodies for pJAK1 (Cell Signaling Technology #3331, RRID: AB_2265057), pJAK2 (Cell Signaling Technology #3771, RRID: AB_330403), p STAT1 (Tyr701; BD #612233, RRID: AB_399556), pSTAT5 (Cell Signaling Technology #9359, RRID: AB_823649), STAT1 (BD #610185, RRID: AB_397584), pSHC (Cell Signaling Technology #2434, RRID: AB_10841301), SHC (Upstate Biotechnology, Inc., #06-203, RRID: AB_310070), pβcY593 (87 (link)), βc (84 (link)), and Actin (Chemicon International, Inc., #MAB1501, RRID: AB_2223041) and goat anti-rabbit (Invitrogen #31463, RRID: AB_228333) or goat anti-mouse (Invitrogen #31437, RRID: AB_228295) IgG Fc secondary antibodies. Immunoblots were analyzed using chemiluminescence on a Bio-Rad Chemidoc Touch.

Kan W.L., Dhagat U., Kaufmann K.B., Hercus T.R., Nero T.L., Zeng A.G., Toubia J., Barry E.F., Broughton S.E., Gomez G.A., Benard B.A., Dottore M., Cheung Tung Shing K.S., Boutzen H., Samaraweera S.E., Simpson K.J., Jin L., Goodall G.J., Begley C.G., Thomas D., Ekert P.G., Tvorogov D., D'Andrea R.J., Dick J.E., Parker M.W, & Lopez A.F. (2023). Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia. Cancer Discovery, 13(8), 1922-1947.

+ Open protocol

+ Expand

Immunoblot Analysis of STAT Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Immunoblots were prepared as described^{41 (link)} using Abs for STAT3 (79D7, #4904), pSTAT3 (Y705, #9145), STAT5 (#9363), pSTAT5 (Y694, 9351), p STAT1 (Y701, #9171), β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA), or STAT1 (#610185) (BD Transduction Labs, San Jose, CA, USA). Following incubation with horseradish peroxidase-conjugated secondary Ab, immune complexes were detected via SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Ware M.B., Zaidi M.Y., Yang J., Turgeon M.K., Krasinskas A., Mace T.A., Keenan K., Farren M.R., Ruggieri A.N., Li Y., Zhang C., Chen Z., Young G.S., Elnaggar O., Che Z., Maithel S.K., Bekaii-Saab T., El-Rayes B, & Lesinski G.B. (2020). Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease. British Journal of Cancer, 123(9), 1377-1386.

+ Open protocol

+ Expand

Western Blotting of Phosphorylated STAT Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

One snap-frozen proliferating and one involuted IH tissue samples from the original cohort of patients used for DAB IHC staining were used for Western blotting (WB). These tissue samples were processed as previously described (3 (link)) and transferred to nitrocellulose membranes (Thermo Scientific) using an iBlot 2 (Thermo Scientific). The membranes were blocked for 90 min at 4°C in 1x iBind™ Flex Solution (Thermo Scientific) and probed using an iBind™ Flex device (Thermo Scientific) with the following primary antibodies: pSTAT1 (1:1,000; cat# 9167, Cell Signaling Technology, Danvers, MA, USA), pSTAT3 (1:1,000; cat# 9145, Cell Signaling Technology), pSTAT5 (1:1000; cat# 9314, Cell Signaling Technology) and β-actin (1:2,000; cat# ab8226, Thermo Scientific). Detection and imaging of the blots were undertaken as routinely done in our laboratory (3 (link)). Human tonsil, mouse lung, and human liver total protein extracts were used as the positive controls for pSTAT1, pSTAT3, and pSTAT5, respectively.

Sulzberger L., Tan E.M., Davis P.F., Brasch H.D., Tan S.T, & Itinteang T. (2018). Phosphorylated Forms of STAT1, STAT3 and STAT5 Are Expressed in Proliferating but Not Involuted Infantile Hemangioma. Frontiers in Surgery, 5, 31.

+ Open protocol

+ Expand

Temporal Signaling Dynamics in C2C12 Myotubes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Four-day differentiated C2C12 myotubes were treated at the time points indicated, washed twice in 1X PBS (Thermo Fisher Scientific), and lysed in radioimmunoprecipitation assay (RIPA) buffer (MilliporeSigma) on ice for 30 minutes. Cells were then scraped into 1.7mL Eppendorf tubes, centrifuged at 9000× g for 10 minat 4 °C, and supernatant collected. Protein lysates were quantified by BCA assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein were separated by SDS-PAGE (4%–15% TGX stain-free gel Bio-Rad, Hercules, CA, USA), and proteins transferred using the trans-blot turbo transfer system (Bio-Rad). The nitrocellulose membranes were probed with the following antibodies from Cell Signaling (Danvers, MA, USA), according to established Western blot protocols: p-STAT3 (#9145), STAT3 (#9139), p-STAT5 (#9351), STAT5 (#94205), p-SMAD2 (#3104), p-SMAD3 (#9520), SMAD2/3 (#8685), p-ERK1/2 (#4370), ERK1/2 (#4695), IκBα (#4814), p-p38 (#9216), p38 (#9212), and GAPDH (#2118). The secondary signal was quantified by fluorescence using a LI-COR Odyssey^® imager (LI-COR Biosciences, Lincoln, NE, USA), and the signal was normalized to a GAPDH loading control.

Callaway C.S., Delitto A.E., D’Lugos A.C., Patel R., Nosacka R.L., Delitto D., Deyhle M.R., Trevino J.G., Judge S.M, & Judge A.R. (2019). IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy. Cancers, 11(12), 1863.

+ Open protocol

+ Expand

Whole Cell Protein Extraction and Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

For whole cell protein extracts, cells were collected and lysed with RIPA buffer (Boston Bioproducts, Ashland, MA). Protein was quantitated using the BCA assay (Pierce, Rockford, IL). Lysates were electrophoretically separated using 12% Tris HCl gels (Biorad, Hercules, CA) and transferred to nitrocellulose membranes. Western blotting was performed using antibodies specific for the following proteins: p STAT5, STAT5, pAkt, Akt, p56lck, phosphor-p56lck, p Erk1/2, Erk1/2, p FoxO1/FoxO3a, FoxO1, GATA-1 (all from Cell Signaling Technologies, Danvers, MA), NFATc1, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP. Average densitometric ratio was calculated for each blot by normalizing to an actin loading control using Image J software.

Grishkan I.V., Tosi D.M., Bowman M.D., Harary M., Calabresi P.A, & Gocke A.R. (2015). Antigenic stimulation of Kv1.3-deficient helper T cells gives rise to a population of Foxp3-independent T cells with suppressive properties. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1399-1407.

+ Open protocol

+ Expand

Quantitative Western Blot Analysis of Signaling Pathways

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blot was performed as previously described (4 (link)). Antibodies against FAK (cat#13009s), p-STAT5
(cat#9351s), STAT5 (cat#9363s), p-AKT (cat#4060s), AKT (cat#2920s), BCL-XL
(cat#2764s), p-ERK (cat#4370s), 4EBP1 (cat#9644), and p-4EBP1 (cat#2855) were
purchased from Cell Signaling Technology (Danvers, MA). Antibodies against MCL-1
(cat#sc-819) and BCL-2 (cat#M0887) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) and DAKO (Carpentaria, CA), respectively. β-actin or
α-tubulin was used as a loading control. Signals were detected using the
Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) and quantified
using Odyssey software (version 3.0, LI-COR Biosciences).

Wang X., Mak P.Y., Mu H., Tao W., Rao A., Visweswaran R., Ruvolo V., Pachter J.A., Weaver D.T., Andreeff M., Xu B, & Carter B.Z. (2020). Combinatorial inhibition of focal adhesion kinase and BCL-2 enhances anti-leukemia activity of venetoclax in acute myeloid leukemia. Molecular cancer therapeutics, 19(8), 1636-1648.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Targets

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blots were performed as previously described [21] (link), using the following antibodies detecting: Akt (cat.# 4691, Cell signalling, dilution 1:1000), Dnmt1 (cat.# ab188453, Abcam; dilution 1:500), Dnmt3a (cat.# SC-20703, Santa Cruz; dilution 1:1000), Dnmt3b (cat.# PA1–884, Thermo Fisher; dilution 1:1000), Stat3 (cat.# SC-8019, Santa Cruz; dilution 1:500), P-Stat3 (cat.# 9145, Cell signalling, dilution 1:1000), Stat5 (cat.# 25656, Cell signalling, dilution 1:1000), P-Stat5 (cat.# 9359, Cell signalling, dilution 1:1000), mouse c-Met (cat.# SC-8057; Santa Cruz; dilution 1:500), c-Myc (cat.# SC-40, Santa Cruz; dilution 1:1000), Hsc-70 (cat.# SC-7298, Santa Cruz; dilution 1:10000), Lin28b (cat.# SC-293120, Santa Cruz; dilution 1:500), h-Ras (cat.# SC-520, Santa Cruz, dilution 1:1000) and β-actin (cat.# SC-130657, Santa Cruz; dilution 1:1000).

Lopusna K., Nowialis P., Opavska J., Abraham A., Riva A, & Opavsky R. (2021). Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling. EBioMedicine, 63, 103191.

+ Open protocol

+ Expand

Western Blot Analysis of Signaling Proteins

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in RIPA buffer containing protease inhibitors and protein lysates were subjected to SDS–PAGE and immunoblot analysis as previously described [34 (link)]. Antibodies used include pSTAT5 (Cell Signaling #9314, 1:1000), total STAT5 (Cell Signaling #9363, 1:1000), β-tubulin (Cell Signaling # 2146S, 1:1000), pFAK Y397 (Cell Signaling, # 3283S, 1:1000), and total FAK (Cell Signaling #3285S, 1:1000).

Jesser E.A., Brady N.J., Huggins D.N., Witschen P.M., O’Connor C.H, & Schwertfeger K.L. (2021). STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment. Breast Cancer Research : BCR, 23, 104.

+ Open protocol

+ Expand

Protein Isolation and Immunoblotting from Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein from snap frozen tissues was isolated as before[49 (link)]. Protein from cell pellets was isolated by pipetting in NP-40 cell lysis buffer (Life Technologies) containing 1% PMSF, incubating on ice for 30 minutes, and removing cleared lysate. Antibodies were purchased from Cell Signaling Technology (p-KIT, Y719, #3391; KIT, D13A2, #3074; p-AKT, Ser473, #4060; AKT, 11E7, #4685; p-S6, S235/236, #2211; S6, 5G10, #2217; p-ERK, T202/Y204, #4370; ERK1/2, #4695; p-STAT5, Tyr694, #9359; STAT5, D2O6Y, #94205; p-STAT3, Y705, #9145; STAT3, 79D7, #4904; p-Tyrosine, p-Tyr-1000, #8954; GAPDH, #5174).

Zhang J.Q., Bosbach B., Loo J.K., Vitiello G.A., Zeng S., Seifert A.M., Medina B.D., Param N.J., Maltbaek J.H., Rossi F., Antonescu C.R., Besmer P, & DeMatteo R.P. (2020). The V654A second-site KIT mutation increases tumor oncogenesis and STAT activation in a mouse model of gastrointestinal stromal tumor. Oncogene, 39(49), 7153-7165.

+ Open protocol

+ Expand

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Whole-cell Brij protein lysis, nuclear and cytosolic protein extractions, and Western-blot analyses were performed as described [46] (link), [65] (link). Antibodies used for detection of the respective proteins and their relevant dilutions were: pSTAT5 (#9351, Cell Signaling Technology; 1∶1000), STAT5A (L-20, sc-1081, Santa-Cruz Biotechnology; 1∶1000), STAT5B (G-2, sc-1656, Santa-Cruz Biotechnology; 1∶200), STAT5A+B (C-17, sc-835, Santa-Cruz Biotechnology; 1∶1000), pJAK2 (Cell Signaling Technology, #3771; 1∶200), JAK2 (#3230, Cell Signaling Technology; 1∶500), α-tubulin (DM1A, sc-32293, Santa-Cruz Biotechnology; 1∶200), Anti-Rabbit IgG-Peroxidase (SIGMA A0545; 1∶10,000), Anti-Mouse IgG-Peroxidase (SIGMA A8924; 1∶10,000). Apparent molecular weight of detected proteins was as predicted by the antibody manufacturers, i.e. 125 kDa for JAK2, 90 kDa for STAT5 (with STAT5A running slightly slower than STAT5B in SDS-PAGE, as confirmed by the STAT5A+B immunoblots) and 55 kDa for α-tubulin. Chemoluminescence detection was performed using Amersham ECL Prime (RPN2232, GE Healthcare Life Sciences) or SuperSignal West Femto (34095, Thermo Fisher Scientific) for weaker signals, and images were captured on an ImageQuant LAS 4000 mini imaging system (GE Healthcare Life Sciences). Immunoblots shown are representative of at least three independent experiments.

Pinz S., Unser S., Brueggemann S., Besl E., Al-Rifai N., Petkes H., Amslinger S, & Rascle A. (2014). The Synthetic α-Bromo-2′,3,4,4′-Tetramethoxychalcone (α-Br-TMC) Inhibits the JAK/STAT Signaling Pathway. PLoS ONE, 9(3), e90275.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!