NK cells were harvested after 14 hour incubation with rIL-2 and lysed with protein lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktail PhosSTOP (Roche Diagnostics, Rotkreuz, Switzerland). Cell lysates were loaded onto SDS-PAGE and transferred to (PDVF) membrane (Gelman, Arbor, MI). This membrane was blocked with 5% skim milk in TBST overnight at 4°C and probed with anti-mouse phospho-Stat3 (Tyr 705; 79/86 kDa) or anti-Stat3 (92 kDa, Cell Signaling Technology, Danvers, MA, USA). After incubation with HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), immunoreactive proteins were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). Band densitometry was performed using LabWorks (BioImaging Systems, UVP, Cambridge, UK).
Mouse anti phospho stat3
Manufactured by Cell Signaling Technology
Sourced in United States
Mouse anti-Phospho-Stat3 is a primary antibody that detects phosphorylated signal transducer and activator of transcription 3 (Stat3) protein. Stat3 is a key transcription factor involved in various cellular processes. This antibody can be used to study the activation and regulation of Stat3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti stat3, by Cell Signaling Technology (2 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Phosstop, by Roche (1 mentions)
Anti mouse phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Wuhan Servicebio Technology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated anti mouse or anti rabbit secondary antibody, by Merck Group (1 mentions)
Rabbit anti phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Anti actin, by Merck Group (1 mentions)
Rabbit anti phospho stat3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Ab108252, by Abcam (1 mentions)
5 protocols using mouse anti phospho stat3
1
Stat3 Activation in NK Cells
2
Western Blot Analysis of STAT3 and NF-κB Signaling
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BMDC whole cell lysates were generated using RIPA buffer containing PMSF and proteins were separated in 10% SDS-PAGE gels and electro-transferred to PVDF membranes and used for Western blot analysis. Primary antibodies used for Western blotting were anti-mouse β-actin (servicebio, Wuhan, China), anti-mouse STAT3(Cell Signaling Technology, Danvers, MA, USA), anti-mouse phospho-STAT3(Cell Signaling Technology) and anti-mouse phospho-NF-κB p65(Cell Signaling Technology). Protein bands were visualized utilizing HRP-conjugated anti-mouse secondary antibodies coupled with ECL detection.
3
Immunostaining of Phospho-Stat3 in Tissue Sections
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Briefly, tissue sections were removed from cryoprotectant and washed in 0.1 M potassium phosphate-buffered saline (KPBS). Tissue was treated at RT with 0.5% NaOH and 0.5% H2O2 in PBS for 20 min. Following KPBS washes, tissue was treated for 10 min with 0.3% glycine at RT. Tissue was subsequently washed in KPBS and followed by a treatment with 0.03% sodium dodecyl sulphate (SDS) in dH2O for 10 min at RT. After KPBS washes, tissue was pre-incubated in blocking buffer [4% normal horse serum + 0.4% Triton X-100 + 1% bovine serum albumin (BSA) in PBS] for 1 h at RT, followed by incubation with mouse anti-Phospho-Stat3 (1:1000, #4113, Cell Signaling, Beverly, MA) in blocking buffer for 1 h at RT and then overnight at 4 °C. Following washes in KPBS, tissue was incubated for 2 h in secondary antibody, Alexa Fluor™ 594 (1:200, Invitrogen, #A21203). Final KPBS washes were performed, and tissue was mounted and imaged.
4
BMP-6 and IL-6 Signaling in HUH7 Cells
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HUH7 cells (1.5×105 per well) were seeded onto 6-well plates and the day after the culture medium was exchanged to FBS-free medium. After 12 hours the cells were treated with increasing doses of BMP-6 (60; 200; 800 ng/mL; R&D Systems) and/or IL-6 (2; 4; 6; 25 ng/mL; R&D Systems) for 12 hours and then harvested for protein analysis. Cells were washed twice in ice cold Dulbecco's phosphate-buffered saline (PBS). Cell pellets were lysed in ice-cold NET buffer (1% Triton X-100 (v/v), 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 20 mM NaF, 1 mM Na3VO4) supplemented with 1× Complete Mini Protease Inhibitor Mixture (Complete Mini,Roche Applied Science). The protein concentration was measured using the BCA (bicinchoninic acid) Protein Assay (Pierce). Protein lysates (15 µg) were subjected to 10% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman) for protein immunodetection using rabbit anti-phospho SMAD 1/5/8 (Cell Signaling #9511), mouse anti-phospho STAT3 (Cell Signaling, #9138) and mouse anti-actin (Sigma Aldrich, A2228). Blots were then incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma Aldrich) and then subjected to chemiluminescence (Amersham Biosciences, ECL Plus). For the densitometric analysis the resulting bands were digitalized and quantified using the NIH Image J software (rsb.info.nih.gov/nih-image/)
5
ACE2 and STAT3 Pathway Profiling
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The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay. 6-OAP was obtained from Sigma-Aldrich, St. Louis, MO, USA, and medicinal plant CM was bought from Beijing Tong Ren Tang Group (Beijing, China). For preparation of CM, the herb (30 g) was soaked in 400 mL water for 30 min, decocted with strong fire to boiling for 15 min and then simmered for 20 min. The decoction was centrifuged to remove insoluble ingredients and filtered with a 0.22 µm pore-size filter, and the sterilized CM was aliquoted and stored at −30 °C.
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