Protein samples were extracted from whole heart homogenates (n = 6/group, chosen randomly) and ADSCs as previously described [21 (link)]. Primary antibody against tumour necrosis factor-α (TNF-α) (R&D, Minneapolis, MN, USA), Akt1/2/3 (Santa Cruze, Santa Cruz, CA, USA), phospho-Akt (Santa Cruze), ERK (Cell Signaling), phosphor-ERK (Cell Signaling), signal transducers and activators of transcription 3 (STAT3; Cell Signaling), phosphor- STAT3 (Cell Signaling), Bcl-2 (Santa Cruze), Bax (Santa Cruze) and horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) were used according to manufacturers’ instructions. GAPDH and β-actin antibody (Cell Signaling Technology) was used to evaluate the amount of protein loaded in each sample (detailed in the supplementary material).
Phosphor stat3
Manufactured by Cell Signaling Technology
Sourced in United States
Phosphor-STAT3 is a lab equipment product that detects the phosphorylated form of the STAT3 protein. STAT3 is a transcription factor that plays a role in cellular signaling pathways. The Phosphor-STAT3 product is used to measure the activation state of STAT3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (9 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Phosphor akt, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Phosphor erk, by Cell Signaling Technology (2 mentions)
Phosphor p65, by Cell Signaling Technology (2 mentions)
Total stat3, by Cell Signaling Technology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Abcam (2 mentions)
Phosphor erk1 2, by Cell Signaling Technology (2 mentions)
Phosphor mtor, by Cell Signaling Technology (2 mentions)
Phosphor p70s6k, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Phosphor jak1, by Cell Signaling Technology (2 mentions)
Phosphor jak2, by Cell Signaling Technology (2 mentions)
Akt1 2 3, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Phospho akt, by Santa Cruz Biotechnology (1 mentions)
20 protocols using phosphor stat3
1
Protein Expression Analysis of Heart and ADSC
2
Western Blot Analysis of Stem Cell Signaling
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Myocardium tissues and AD-MSCs were harvested for Western Blot following standard protocol. Samples consisting of 50 μg total protein were loaded onto an SDS-PAGE gel (Beyotime, China) and transferred electrophoretically to nitrocellulose membranes (LC2000, Invitrogen, USA). After blocked with 5% bovine serum albumin in PBS, the membranes were incubated with the appropriate primary antibody against Akt, phospho-Akt, ERK, phosphor-Erk, Stat3, phosphor- Stat3, all from Cell Signaling Technology, Danvers, MA,USA) overnight. The next day, the blots were washed and incubated in the appropriate secondary antibodies (Abcam, Cambridge, MA, USA) at room temperature for 1 h. Immunoreactivity was then detected by sequential incubation with HRP-conjugated antibodies and enzymatic chemiluminescence. Quantitative analysis was performed using QuantiOne imaging software (Bio-Rad, USA) to assess the integrated optical density (IOD) of each band.
3
Immunohistochemical Analysis of Kidney Samples
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Paraffin-embedded sections of mouse and human kidneys were incubated with primary antibodies and then with biotinylated second antibodies, followed by incubation with an avidin–biotin–peroxidase complex; they were developed using substrate provided by Vector Laboratories. The human biopsy and nephrectomy samples were obtained under a protocol approved by the institutional review board of Icahn School of Medicine at Mount Sinai (HS#: 11–02130). The following antibodies were used in the study: SIRT1 (cat. no. 07131; Millipore), acetyl-STAT3 (cat. no. 2523; Cell Signaling Technology), phosphor-STAT3 (cat. no. 9145; Cell Signaling Technology), acetyl-p65 (cat. no. 3045; Cell Signaling Technology), phosphor-p65 (cat. no. 3033; Cell Signaling Technology), and synaptopodin (provided courtesy of Dr. Peter Mundel). The dilutions of both acetyl-p65 and acetyl-STAT3 antibodies for immunostaining were 1:50. Negative controls for acetyl-STAT3 and acetyl-p65 were obtained by preabsorption with a synthetic acetylated peptide corresponding to residues surrounding Lys310 of p65 or Lys685 of STAT3.
4
Western Blot Protein Analysis
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Western blots were performed as previously described [24 (link)]. The primary antibodies used were Hes1 (Millipore, Billerica, MA), MMP14 (Abcam, Cambridge, UK), STAT3, phosphor-STAT3 (Cell Signaling Technology, Danvers, MA), Flag and Actin (Sigma-Aldrich). Membranes were then incubated with a specific primary antibody overnight, washed, then incubated with an appropriate secondary antibody conjugated to horseradish peroxidase, and developed using ECL (PerkinElmer Life Sciences, Waltham, MA).
5
Western Blot Analysis of Phosphorylated Signaling Proteins
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The cellular proteins were extracted by lysing cells in a RIPA buffer containing 1mM Phenylmethylsulfonyl Fluoride (PMSF). The protein samples were separated by SDS-PAGE electrophoresis, and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with bovine serum albumin (5%) for 1 h at room temperature under slight shaking, the membranes were incubated with primary antibodies (1:500) at 4 °C for 12 h and then blotted with secondary antibody for 2 h at room temperature. Then the bands were stained with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA). In this study, the cells including M2-M and TAMs were treated with 50 μg/mL IAPS-2 for 24 h, then the expression of phosphorylated P65 (p-P65), total P65, phosphorylated STAT1 (p-STAT2), total STAT1 (T-STAT1), phosphorylated STAT3 (p-STAT3) and total STAT3 (T-STAT3) in both cells were evaluated by Western blot. The corresponding antibodies, including phosphor-P65, Total-P65, phosphor-STAT1, Total-STAT1, phosphor-STAT3, Total-STAT3 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Osteosarcoma Cell Line Regulation
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The U2OS and MG-63 osteosarcoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were routinely cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% FBS (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin in 5% CO2 at 37°C. Rabbit anti-STAT3, phosphor-STAT3, rabbit anti-ERK1/2, phosphor-ERK1/2, rabbit anti-p38, phosphor-p38, rabbit anti-MMP-2, rabbit anti-MMP-9 and rabbit anti-Snail antibodies were all purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-E-cadherin, vimentin, N-cadherin, fibronectin and rabbit anti-MMP-7 antibodies were purchased from Abcam (Cambridge, UK). Alexa Fluor 488-conjugated goat anti-rabbit IgG was purchased from Invitrogen Life Technologies. IL-6 was purchased from PeproTech (Rocky Hill, NJ, USA). Irisin was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). WP1066, a selective inhibitor of STAT3, was purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Immunoblotting for Signaling Proteins
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Antibodies against phosphor-AMPKα, AMPKα, phosphor-ACC, ACC, phosphor-mTOR, mTOR, phosphor-p70S6K, p70S6K, phosphor-4E-BP1, 4E-BP1, phorphor-NFκB p65, NFκB p65, IκBα, COX2, phosphor-STAT3, STAT3, Mcl-1, Bcl-xL, Bim, Bak, Bax, Bid, Puma and Bad were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and Protein A/G Agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Metformin and aspirin as well as interleukin-6 (IL-6) were purchased from Sigma-Aldrich (St Louis, MO, USA). AZD-8055 was purchase from Invitrogen (Carlsbad, CA, USA).
8
Protein Quantification and Western Blotting
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Samples used for this assay were collected from whole-cell lysates. A coomassie assay (Pierce, Rockford, IL) was used to quantify the total protein concentration. Identical amounts of protein were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and electro-transferred to polyvinylidene fluoride membranes. The following primary antibodies were used in this study: Bim, Noxa, PUMA (ProSci, Poway, CA); Mcl-1, caspase 3, and caspase 8 (BD Biosciences); Bcl-2 (DAKO, Carpinteria, CA); cyclin E1 (BD Biosciences), Bax (R&D Systems), Actin (Santa Cruz Biotechnology), Bcl-xL, Bad, cyclin D1, cyclin A1, STAT3, and phosphor-STAT3 (Cell Signaling Technology).
9
Western Blot Analysis of Cellular Proteins
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For Western blotting assay, cellular proteins were obtained by lysing cells in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amounts of samples were subjected to SDS-PAGE electrophoresis and then transferred to PVDF membranes (Bio-Rad, USA). After blocking with skimmed milk or bovine serum albumin (5%) for 1.5 h at room temperature with gentle shaking, the membranes were blotted with primary antibodies (1 : 1000) overnight at 4 °C and then exposed to secondary antibody for 2 hours at room temperature. The bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA). The corresponding antibodies including phosphor-STAT3, Total-STAT3 and GAPDH were obtained from cell signaling technology (Danvers, MA, USA).
10
Adiponectin Signaling in Cell Models
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Recombinant rat globular adiponectin (gAcrp) was purchased from BioVision (Mountain View, CA). Adiponectin was produced using bacteria (Escherichia coli). Dulbecco’s modified eagle medium (DMEM), Trypsin-EDTA, collagenase, penicillin/streptomycin, Opti-MEM Reduced-Serum Medium and fetal bovine serum (FBS) were all obtained from Gibco Laboratories (Grand Island, NY). Antibodies for phosphor-AMPK, whole AMPK, phosphor-Akt, whole Akt, phosphor-STAT3, whole STAT3, APPL1, and NF-κB p65 were acquired from Cell Signaling Technology (Beverly, MA). The antibody for AdipoR1 was from Abcam (Cambridge, UK). All target siRNAs and Cy3-labeled non-silencing siRNA (NS siRNA) were purchased from Ribobio Co. (Guangzhou, China), and TransIT-TKO transfection reagent was acquired from Mirus Bio Corporation (Madison, WI). AngII, compound C, BrdU and LY294002 were acquired from Sigma-Aldrich (St. Louis, MO). Finally, Ro31-8220 was obtained from Millipore (Billerica, MA). All reagents were of analytical grade.
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