Unless otherwise stated, all chemicals, including aspirin (ASA), LPS (Escherichia coli 055:B5), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and mouse monoclonal anti-β-actin, were obtained from Sigma Chemical Co., St. Louis, MO, USA. A BCA protein assay kit and a Revert Aid First Strand cDNA Synthesis Kit were purchased from Thermo Scientific, Waltham, MA, USA, and TRIZOL Reagent from Life technologies, Carlsbad, CA, USA. Rabbit monoclonal anti-phospho-JAK2, rabbit monoclonal anti-JAK2, rabbit polyclonal anti-phospho-STAT3, mouse monoclonal anti-STAT3, rabbit monoclonal anti-phospho-P65, and rabbit monoclonal anti-P65 antibodies were supplied by Cell Signaling Technology, Inc., Danvers, MA, USA, and mouse monoclonal anti-IRP1 was from Abcam, San Francisco, CA, USA. Goat anti-rabbit and anti-mouse IRDye 800CW secondary antibodies were bought from LI-COR bio sciences, Lincoln, NE, USA. The Health Department of Hong Kong and Shanghai Government and the Animal Research Ethics Committee of The Chinese University of Hong Kong (the project identification code: GRF14106914, 1 January 2015) and Fudan University (the project identification code: NSFC31271132, 1 January 2013; NSFC31330035, 1 January 2014) approved the experimental procedures of this study.
Mouse monoclonal anti stat3
Manufactured by Cell Signaling Technology
Sourced in United States
Mouse monoclonal anti-STAT3 is a laboratory reagent used to detect and study the STAT3 protein. It is a purified mouse monoclonal antibody that specifically binds to STAT3.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit monoclonal anti jak2, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti irp1, by Abcam (2 mentions)
Mouse monoclonal anti β actin, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Rabbit polyclonal anti phospho stat3, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
α actin, by Merck Group (1 mentions)
Nupage bis tris, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Rabbit polyclonal anti pml, by Fortis Life Sciences (1 mentions)
Rabbit polyclonal anti sox9, by Merck Group (1 mentions)
Rabbit polyclonal anti sox2, by Merck Group (1 mentions)
Rabbit monoclonal anti phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Anti eed, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti myc, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti h3k27me3, by Merck Group (1 mentions)
11 protocols using mouse monoclonal anti stat3
1
Investigating JAK2/STAT3 Signaling Pathways
2
Immunoblotting Protocol for Protein Analysis
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For immunoblotting studies, 20 μg of proteins from each sample were resolved on 4–12% polyacrylamide gel electrophoresis NuPAGE Bis-Tris (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes. Rabbit polyclonal anti-Phospho-EGFR (Tyr1068), -Phospho-EGFR (tyr1173), -Phospho-Akt (Ser473) -Akt, -Caspase-3 (8G10) and -Phospho-Stat3 (Ser727) and mouse monoclonal anti-STAT3 were purchased from Cell Signaling (Beverly, MA, USA). Mouse monoclonal anti-PTEN was purchased from BD (Franklin Lake, NJ, USA), mouse monoclonal anti-Phospho-ERK clone (E-4), rabbit polyclonal anti-EGFR (1005), -Bcl xL (H5), -Erk and -Notch3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and mouse monoclonal β-tubulin and α-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Peroxidase-conjugated secondary antibodies were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).
3
Immunoblot Analysis of Transcription Factors
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Immunoblots were performed as previously described [50 (link)]. The following primary antibodies were used: rabbit polyclonal anti-SOX9 (1:2000 dilution; Millipore, Burlington, MA, USA), mouse monoclonal anti-STAT3 (1:1000 dilution; Cell Signaling), rabbit monoclonal anti-phospho STAT3 (Tyr705) (1:1000 dilution; Cell Signaling), rabbit polyclonal anti-PML (1:1000 dilution; Bethyl laboratories, Montgomery, TX, USA), rabbit polyclonal anti-SOX2 (1:500 dilution; Millipore), and mouse monoclonal anti-β actin (1:2000 dilution; Sigma-Aldrich). Primary antibodies were detected with horseradish peroxidase (HRP)-linked antibodies: Goat anti-rabbit or goat anti-mouse (Santa Cruz Biotechnology, Dallas, TX, USA). Protein detection was performed using the NOVEX® ECL system (Invitrogen, Carlsbad, CA, USA).
4
Immunophenotyping of Transcription Factors
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Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).
5
Molecular Mechanisms of Iron Homeostasis
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Unless otherwise stated, all chemicals, including Lipopolysaccharides (LPS) and mouse monoclonal anti-β-actin antibody, were obtained from Sigma-Aldrich Chemical Co., St.
Louis, MO, USA. The mouse anti-ratTfR1 was purchased from Invitrogen, Carlsbad, CA, USA, rabbit polyclonal anti-mouse DMT1 from Alpha Diagnostic International Company, San Antonio, TX, USA, rabbit polyclonal anti-mouse Fpn1 from Novus Biologicals, Littleton, CO, USA; rabbit polyclonal anti-Ft-L from Proteintech, Chicago, IL, USA; mouse monoclonal anti-IRP1 from Abcam, San Francisco, CA, USA; rabbit monoclonal anti-p-JAK2 (phospho-Janus Kinase 2), rabbit monoclonal anti-JAK2, rabbit polyclonal anti-phospho-STAT3 (Tyr705) and mouse monoclonal anti-STAT3 both from Cell Signaling
Louis, MO, USA. The mouse anti-ratTfR1 was purchased from Invitrogen, Carlsbad, CA, USA, rabbit polyclonal anti-mouse DMT1 from Alpha Diagnostic International Company, San Antonio, TX, USA, rabbit polyclonal anti-mouse Fpn1 from Novus Biologicals, Littleton, CO, USA; rabbit polyclonal anti-Ft-L from Proteintech, Chicago, IL, USA; mouse monoclonal anti-IRP1 from Abcam, San Francisco, CA, USA; rabbit monoclonal anti-p-JAK2 (phospho-Janus Kinase 2), rabbit monoclonal anti-JAK2, rabbit polyclonal anti-phospho-STAT3 (Tyr705) and mouse monoclonal anti-STAT3 both from Cell Signaling
6
Immunohistochemical Detection of STAT3 in Cells
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Cells were fixed in a 24-well plate with 4% PFA in PBS (pH 7.4) for 30 min at room temperature (RT). After fixation cells were washed five times with PBS (5 min each), blocked and permeabilised with 5% normal goat serum and 0.1% saponin in PBS for 30 min, and then incubated with primary antibody anti-STAT3 mouse monoclonal (9139, Cell Signaling Technology) overnight at 4°C in PBS 5% normal goat serum and 0.05% saponin. After five washes with PBS (5 min each), cells were incubated with HRP-conjugated Fab fragments of the secondary antibody for 2 h at RT. After five washes, cells were incubated in the DAB solution (0.01 g DAB in 20 ml TRIS-HCl buffer plus 30% H2O2 solution added immediately before use). The samples were then postfixed with 1% osmium tetroxide plus potassium ferrocyanide 1% in 0.1 M sodium cacodylate buffer for 1 h at 4°C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin sections (60-70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate and viewed with a Tecnai G2 (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System) digital camera.
7
Immunoblotting of Mouse ESC Mitochondria
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Immunoblotting was performed as previously described in Carbognin et al. (2016) (link). The following antibodies were used: anti-STAT3 mouse monoclonal (9139, Cell Signaling Technology, 1:1000), anti-GAPDH mouse monoclonal (MAB374, Millipore, 1:1000), anti-VDAC1 rabbit polyclonal (ab15895, Abcam, 1:1000), anti-Lamin (sc-6217, Santa Cruz Biotechnology, 1:1000), anti-βActin mouse monoclonal (MA1-744, Invitrogen, 1:10,000) and anti-pSTAT3 S727 rabbit monoclonal (9134, Cell Signaling Technology). Mitochondria from mouse ESCs were isolated using Mitochondria Isolation Kit (89874, Thermo Fisher Scientific).
8
Immunofluorescence Staining of ESCs
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ESCs were grown and transfected as previously described by Carbognin et al. (2016) (link). For IF, ESCs were fixed for 10 min in cold methanol at −20°C, washed in TBS, permeabilised for 10 min with TBST (Tris-buffered saline/Tween 20)+0.3% Triton X-100 at RT, and blocked for 45 min in TBS+3% goat serum at RT. The cells were incubated overnight at 4°C with primary antibodies anti-STAT3 mouse monoclonal (9139, Cell Signaling Technology, 1:100) and anti-ATAD3A rabbit monoclonal (224485, AB-Biotechnologies, 1:100). After washing with TBS, the cells were incubated with secondary antibodies (Alexa Fluor 568, raised in donkey anti-mouse IgG, diluted 1:500, Thermo Fisher, A-11036; Alexa Fluor 488, raised in goat anti-rabbit IgG, diluted 1:500, Thermo Fisher, A-21206) for 30 min at RT. Cells were mounted with ProLong® Gold Antifade Mountant with DAPI (P36941, Life Technologies) or HOECHST 33342 (62249, Thermo Fisher Scientific) where specified. Images were acquired using a Leica SP2 confocal microscope equipped with a CCD camera.
9
Immunofluorescence Detection of STAT3
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Cells were fixed in a 24 wells plate with 4% Paraformaldehyde in PBS (pH 7.4) for 30 minutes at RT (room temperature). After fixation cells were washed 5 times with PBS (5 minutes each), blocked and permeabilized with 5% normal goat serum and 0.1% saponin in PBS for 30 min, and then incubated with primary antibody anti-STAT3 mouse monoclonal (9139, Cell Signalling) ON at 4°C in PBS 5% normal goat serum and 0.05%
10
Western Blot Analysis of Phosphorylated Proteins
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The nuclear and cytoplasmic fractions were aliquoted (60 μg each) and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corporation) using a wet blotting apparatus (Bio-Rad, Inc., Hercules, CA, USA), and the membrane was incubated in blocking buffer [Tris-buffered saline, Tween 20 (0.05%), and bovine serum albumin (5%) or nonfat milk (5%)] for 1 hour at room temperature, followed by overnight incubation at 4°C in a 1:1,000 dilution of polyclonal rabbit anti-phospho-STAT3 (Tyr705; Cell Signaling Technology, Inc., Danvers, MA, USA), polyclonal rabbit anti-phospho-SHP2 (Tyr580; Millipore Corporation), monoclonal mouse anti-STAT3 (Cell Signaling Technology, Inc.), polyclonal rabbit anti-SHP2 (Millipore Corporation), or monoclonal mouse anti-β actin antibody (Sigma-Aldrich, St. Louis, MO, USA). The membrane was then washed 4 times for 10 minutes in TBS-T. Next, the membrane was incubated in 5% nonfat milk blocking buffer containing a 1:3,000 dilution of horseradish peroxidase–linked goat antirabbit and antimouse IgG (Bio-Rad, Inc.). The washes were repeated, and the membrane was developed with chemiluminescence agent (ECL; Amersham Life Science, Inc., Arlington Heights, IL, USA). The band densities were quantified using ImageJ software (NIH Image, Bethesda, MD, USA).
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