Isolated CD8+ OT-I T cells were cultured in 96-well plates and activated through PMA and ionomycin stimulation or co-culture with SL8-pulsed activated BMDCs. Anti-mouse IL-2 (15 μg/ml, JES6-1A12) was added to cultures at time of plating to restrict IL-2 delivery. After 24 h, recombinant human IL-2 (0.1–100 U/ml) was added to wells for 20 min at 37 °C. Cells were washed, fixed with 4% PFA for 15 min at 20 °C, and washed. Cells were resuspended with cold (−20 °C) MeOH and washed with flow cytometry buffer 4 times. To stain, cells were incubated in PBS containing 2% BSA and anti-phospho-STAT5 (C71E5; Cell Signaling Technology) before a final wash and resuspension in flow cytometry buffer.
Anti phospho stat5
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-STAT5 is a laboratory research reagent used to detect the phosphorylated form of the transcription factor STAT5. It is designed for use in various techniques, such as Western blotting, to study the activation and signaling pathways involving STAT5 in cells.
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Lab products found in correlation
Anti stat5, by Cell Signaling Technology (19 mentions)
Anti phospho stat3, by Cell Signaling Technology (8 mentions)
Anti erk1 2, by Cell Signaling Technology (6 mentions)
Anti phospho jak2, by Cell Signaling Technology (5 mentions)
Anti phospho akt, by Cell Signaling Technology (5 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (5 mentions)
Anti stat5, by Santa Cruz Biotechnology (4 mentions)
Anti jak2, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti phospho erk, by Cell Signaling Technology (4 mentions)
Anti phospho s6, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti β actin, by Merck Group (4 mentions)
Anti c abl, by Cell Signaling Technology (3 mentions)
Anti phospho crkl, by Cell Signaling Technology (3 mentions)
Anti phospho stat1, by Cell Signaling Technology (3 mentions)
Anti erk, by Cell Signaling Technology (3 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
40 protocols using anti phospho stat5
1
Phospho-STAT5 Activation in OT-I T Cells
2
Western Blot Analysis Protocol
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Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to nitrocellulose membranes (Amersham). Membranes were blocked overnight in 5% w/v BSA and incubated with primary antibody for 2 h. Anti-GST-HRP (RPN1236, 1:10000) was from Sigma-Aldrich. Anti-phosphotyrosine antibody (4G10) was obtained from Millipore. Anti-Elongin C antibody (610761, 1:3000) was from BD Biosciences. Anti-p44/42 (9102S, 1:2000), anti-STAT5 (94205; 1:3000), and anti-phospho-STAT5 (9359; 1:2000) were purchased from Cell Signaling. Anti-actin-HRP antibody (C4) was obtained from Santa Cruz (sc-47778 HRP; 1:1000). Anti-Cullin 5 (ab184177), anti-Elongin B antibody (ab154854, 1:2000), and anti-SOCS2 antibody (ab109245, 1:1000) were purchased from Abcam. Antibody binding was visualized with peroxidase-conjugated sheep antirabbit immunoglobulin (Southern Biotech; 4010-05; 1:15000), or sheep antimouse immunoglobulin (GE Healthcare; NA931-1ML; 1:10000) and the enhanced chemiluminescence (ECL) system (Amersham or Millipore).
3
Western Blot Antibody Optimization
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The following primary Abs were used: rabbit polyclonal anti-phospho-Jak2 (Tyr1007/1008), anti-phospho-Stat3 (Tyr705), anti-phospho-Stat5 (Tyr694), anti-phospho-Akt (Ser473), anti-phospho-eIF4E (Ser209), anti-phosp ho-4E-BP1 (Ser65) used at 1/1000 dilution (Cell Signaling Technology), and mouse monoclonal anti-β-actin (1/5000; Sigma-Aldrich; St. Louis, MO) and anti-GAPDH (1/5000; Abcam, Cambridge, MA) antibodies. Goat anti-mouse EPOR (use at 0.2ug/ml) was purchased from R&D, and anti-total 4E-BP1, anti-total Jak2, anti-total eIF4E, and anti-total Akt were from cell Signaling. Secondary Abs included: anti-goat IgG-HRP (1/2500; Santa Cruz, Santa Cruz, CA), anti-rabbit and anti-mouse IgG-HRP (1/2000; Cell Signaling Technology).
4
Evaluation of GM-CSF-induced STAT5 Phosphorylation
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GM-CSF-stimulated phosphorylation of STAT5 in peripheral blood monocytes (PBMC) and granulocytes was evaluated in six patients according to a modified protocol by Suzuki et al. [11 (link)]. Heparinized blood was processed within 24 hours. Mononuclear cells and granulocytes were isolated using a Ficoll-Paque plus gradient (GE Healthcare, Uppsala, Sweden) and Leucosep tubes (Greiner Bio One, Solingen, Germany). Cell suspensions were incubated with or without GM-CSF (Leukine, Berlex, Bayer Healthcare, Seattle, WA; 100 ng/ml, 15 minutes, 37°C). Expression of phosphorylated STAT5 (pSTAT5) was measured by flow cytometry (BD FACSCanto). In a subset of cases, cell lysates were also evaluated by Western blotting using anti-STAT5 (Santa Cruz Biotechnology; Santa Cruz, CA), anti-phospho-STAT5 (Cell Signaling), or anti-beta actin (Santa Cruz) as primary antibodies.
5
Western Blot Analysis of Larval Responses
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At each time point, 30 larvae from control or metronidazole treatment groups were harvested in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, 78830), Protease Inhibitor Cocktail (Promega, G6521), Phosphatase Inhibitor Cocktail 2 (Sigma, P5726), and Phosphatase Inhibitor Cocktail 3 (Sigma, P0044). Western blotting was performed using standard protocols [48 (link)]. Eleven primary antibodies were used in this study: anti-IL-7R (1:1500; Abcam, ab180521), anti-MBP (1:500; Anaspec, #55811, Fermont, CA, USA), anti-Phospho-JAK1 (1:1,000; Cell Signaling Technology, #3331, Danvers, MA, USA), anti-Phospho-JAK3 (1:500; Cell Signaling Technology, #5031), anti-STAT3 (1:500; Santa Cruz Biotechnology, sc-7179, TX, USA), anti-Phospho-STAT3 (1:200; Cell Signaling Technology, #9145), anti-STAT5 (1:500; Santa Cruz Biotechnology, sc-836), anti-Phospho-STAT5 (1:200; Cell Signaling Technology, #9351), anti-BCL2 (1:1000; BD Biosciences, 610538), anti-CASPASE-3/cleaved CASPASE-3 (1:300; Wanleibio, WL02117), and anti-CASPASE-7/cleaved CASPASE-7 (1:500; Wanleibio, WL0181). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:3,000; Millipore, MAB374, Billerica, MA, USA) was used as a loading control.
6
Ubiquitin-mediated protein regulation assay
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Spautin-1 (S7888), Imatinib (S1026), and MG132 (S2619) were from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was from Xcessbio Biosciences, Inc. (San Diego, CA). MTS reagent (G3582) was from Promega Corporation (Madison, WI, USA). Co-IP assay kit (14311D) was from Life Technologies (Carlsbad, CA). Antibodies: anti-Ubiquitin (#3936), anti-K63-Ubiquitin (#12930), anti-K48-Ubiquitin (#12805), anti-USP10 (#8501), anti-USP1 (#8033), anti-USP2 (#8036), anti-USP7 (#4833), anti-USP8 (#11832), anti-USP14 (#11931), anti-USP15 (#66310), anti-USP18 (#4813), anti-UCHL1 (#13179), anti-UCHL3 (#8141), anti-CYLD (#8462), anti-A20 (#5630), anti-SKP2 (#2652), anti-p27 (#3686), anti-FLAG (#14793), anti-HA (#3724), anti-phospho-c-Abl(Y245) (#2861), anti-c-Abl (#2862), anti-phospho-STAT5 (#9359), anti-STAT5 (#25656), anti-phospho-Crkl (#3181) and anti-Crkl (#38710) (Cell Signaling Technology, Beverly, MA, USA); anti-UCHL5 (ab124931), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630) (Bioworld Technology, Inc., Louis Park, MN, USA).
7
Evaluating JAK/STAT Pathway Inhibition
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The effect of JAK1/2 inhibitors ruxolitinib and AZD1480 on JAK2 and STAT5 phosphorylation was evaluated by western blotting. Cells were washed to remove traces of serum and incubated with inhibitor for 90 minutes. Cells were lysed in SDS lysis buffer containing protease inhibitors and separated by SDSPAGE. Antibodies employed were anti-JAK2 (Abcam, Cat.No. ab108596), anti-phospho-JAK2 (Cell Signaling Technology, Cat.No. 3776), anti-STAT5 (Cell Signaling Technology, Cat.No. 94205), anti-phospho-STAT5 (Cell Signaling Technology, Cat.No. 9351) and anti-GAPDH (Cell Signaling Technology, Cat.No. 2118).
8
Murine Ba/F3 Cell Culture Protocol
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Low-passage murine Ba/F3 cells, kindly provided by Connie J. Eaves (Terry Fox Laboratory, British Columbia Cancer Agency and University of British Columbia, Vancouver, BC, Canada), were cultured in RPMI-1640 (Wisent Bio Products, QC, Canada) supplemented with 10% FBS (Wisent Bio Products), 100 units/mL penicillin and streptomycin (Wisent Bio Products, QC, Canada) and 5 ng/ml of recombinant murine IL3 (Peprotech, NJ, USA) at 37 °C in a humidified atmosphere at 5% CO2. Cells infected with lentiviral plasmids were selected by adding puromycin (1 μg/mL) (Wisent Bio Products, QC, Canada) to the cell culture medium. Stable infection of Ba/F3 cells was performed as described previously [27 (link)]. Expression of FLT3 was confirmed by RT-qPCR and Western blot as described elsewhere [28 (link)]. The following antibodies were used: anti-FLT3 (Cell Signaling, #3462, Danvers, MA, USA), anti-phospho ERK1/2 (Cell Signaling, #4370, Danvers, MA, USA), anti-ERK1/2 (Cell Signaling, #4695, Danvers, MA, USA), anti-phospho STAT5 (Cell Signaling, #9359, Danvers, MA, USA) and anti-STAT5a/b (Abcam, EPR16671-40, Cambridge, UK) and anti-GAPDH (sc-32233) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
9
Phospho-STAT5 Activation in OT-I T Cells
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Isolated CD8+ OT-I T cells were cultured in 96-well plates and activated through PMA and ionomycin stimulation or co-culture with SL8-pulsed activated BMDCs. Anti-mouse IL-2 (15 μg/ml, JES6-1A12) was added to cultures at time of plating to restrict IL-2 delivery. After 24 h, recombinant human IL-2 (0.1–100 U/ml) was added to wells for 20 min at 37 °C. Cells were washed, fixed with 4% PFA for 15 min at 20 °C, and washed. Cells were resuspended with cold (−20 °C) MeOH and washed with flow cytometry buffer 4 times. To stain, cells were incubated in PBS containing 2% BSA and anti-phospho-STAT5 (C71E5; Cell Signaling Technology) before a final wash and resuspension in flow cytometry buffer.
10
Phosphorylated STAT5 Detection Assay
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