The total proteins from tissue and cell samples were washed three times with PBS. Then the samples were lysed in RIPA lysis buffer for 30 min. The protein lysates were separated by 10% SDS-PAGE. The specific proteins were transferred on PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 30 min and then washed twice with TBST. The specific proteins were incubated with the different primary antibodies overnight at 4°C. The used primary antibodies were as follows: JAK1 (Ab-1022), p-JAK1 (SAB4300123), JAK2 (Ab-570), p-JAK2 (SAB4301238), p-STAT3 (SAB4300034), Ki67 (SAB4501880) and STAT3 (SAB4502871) (Sigma-Aldrich), cleaved caspase-3 (9654), matrix metalloproteinases [MMP-2 (40994) and MMP-9 (15561)], E-cadherin (14472), N-cadherin (4068), Snail (3879) and β-actin (8457) (Cell Signaling Technology, Beverly, MA, USA). The membranes were washed twice and then incubated with HRP-linked secondary antibody (7075; Cell Signaling Technology) for 1–2 h. Protein signals were visualized using enhanced chemiluminescence reagents (ECL; GE Healthcare, Waukesha, WI, USA).
Stat3
Manufactured by Merck Group
Sourced in United States
STAT3 is a lab equipment product manufactured by Merck Group. It is a protein that functions as a transcription factor, regulating gene expression in response to various cellular stimuli.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Pstat3, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Cleaved caspase 3, by Merck Group (1 mentions)
P jak2, by Merck Group (1 mentions)
E cadherin 14472, by Cell Signaling Technology (1 mentions)
Hrp linked secondary antibody, by Cell Signaling Technology (1 mentions)
Mmp 2 40994, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Anti phospho stat3, by Merck Group (1 mentions)
Anti rabbit igg antibody, by Merck Group (1 mentions)
Anti mouse igg antibody, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Immun star hrp chemiluminescence kit, by Bio-Rad (1 mentions)
Il 1β, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
16 protocols using stat3
1
Western Blot Analysis of Protein Signaling
2
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein and tissue protein extract were prepared and western blotting was performed10 (link)15 . The extracted proteins were transferred to PVDF membranes (Invitrogen, USA) and incubated with primary antibodies i.e. anti-ferritin antibody (1:1000; Sigma-Aldrich, USA) anti-SLC-40A1 antibody (1:5000; Sigma-Aldrich, USA), anti phospho-Stat-3 (1:1000; Sigma-Aldrich, USA), Stat-3 (1:1000; Sigma-Aldrich, USA). Further the blot was incubated with secondary peroxidase-labelled antibodies, anti-rabbit IgG antibody (1:5000; Sigma-Aldrich USA) and anti-mouse IgG antibody (1:1000; Sigma-Aldrich USA) for 1 h. Anti-α-tubulin antibody was used as a loading control. PVDF membrane for immune-reactive band was detected by immunostaining kit (Sigma-Aldrich, USA). Immunoblot were quantified using ImageJ software, and band densities were normalized with the corresponding Tubilin band densities. The blot shown is representative of 3 independent experiments.
3
Western Blot Analysis of Neuroinflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed to measure protein expression of IL-6, TNF-α, IL-1β, JAK2, and STAT3. Hippocampus tissues were homogenized in strong RIPA buffer. The homogenate was allowed to rest on ice for 30 min and then centrifuged at 15,000 rpm for 15 min at 4°C, and the supernatant was collected. The protein concentration of tissue lysates was determined with a BCA protein assay kit. The lysates were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat powdered milk in TBST (Tris-buffered saline containing 0.1% Tween 20) for 1 h at room temperature (RT) and then incubated overnight at 4°C with the following primary antibodies: IL-6 (Abcam); IL-1β (Millipore); TNF-α (Santa Cruz); JAK2 (Sigma-Aldrich), STAT3 (Sigma-Aldrich), β-actin (Abcam). After washing in TBST, the membrane was incubated for 1 h at RT with HRP-conjugated goat anti-rabbit antibody (West Grove), and protein bands were visualized using the Immun-Star™ HRP Chemiluminescence Kit (Bio-Rad). Images of bands were recorded by the ImageQuant LAS 4000 system (GE Healthcare) and the band intensities were quantified using ImageQuant TL software (version 7.0, GE Healthcare). β-Actin was used as the internal loading control.
4
Protein Expression Analysis in ASMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from ASMCs using RIPA lysis buffer (Fermentas, Glen Burnie, MD, U.S.A.). After determination of protein concentration, equal amount of protein extracts was separated by 10–12% SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A.). After incubation with blocking buffer, 5% non-fat milk, for 1 h, primary antibodies against α-SMA (1:500; Abcam, Cambridge, MA, U.S.A.), myocardin (1:500; Sigma–Aldrich, St. Louis, MO, U.S.A.), nuclear factor-κ B (NF-κB) p65 (1:500; Sigma–Aldrich), p-p65 (1:500; Abcam), signal transducer and activator of transcription 3 (STAT3; 1:500; Sigma–Aldrich), p-STAT3 (1:500; Sigma–Aldrich), p-AKT (1:1500; Abcam), AKT (1:2000; Abcam), and β-actin (1:500; Abcam) were added to the incubation system and incubated at 4°C overnight. Then the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000; Abcam) at 37°C for 1 h. The protein bands on the membranes were visualized using ECL reagent (ECL, Pierce, Rockford, IL, U.S.A.). The protein levels were analyzed by detecting the density using ImageJ Software (National Institutes of Health, NIH, Bethesda, MD, U.S.A.).
5
Mogroside V Regulation of STAT3 and Cell Cycle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with mogroside V for 24 h and were lysed at 4 °C in lysis buffer (0.05 M Tris-HCl, pH 7.4, 1% Triton X-100, 0.1 M NaCl, 1% sodium deoxycholate and 0.1% SDS) containing 1 mM phenylmethanesulfonyl fluoride and a protease inhibitor. The insoluble protein lysate was removed by centrifugation. About 15 μg of protein from the cell lysates were separated by 12% SDS–PAGE and were subsequently transferred onto a polyvinylidene fluoride membrane. The following primary antibodies were used for western blot analysis: STAT3 (1:1000 dilution), phospho-(p)-STAT3, CDKN1A, CDKN1B (p27), BCL2 and β-actin (all from Sigma-Aldrich). After incubation with the appropriate secondary antibodies (all from Sigma-Aldrich), the membranes were developed with ECL plus (ECL, Amersham Biosciences, GE Healthcare, Pittsburgh, PA, USA). Densitometric measurements of the protein bands were performed using the GS-800 imaging densitometer (Bio-Rad, Hercules, CA, USA).
6
SDS-PAGE and Western Blotting Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS page and western blot of NP-40 lysed material was performed using a standard protocol and documented by ponceau S staining. Primary antibodies (obtained from Cell Signaling Technology Inc. Danvers, MA, USA: pan-actin (D18C11), phospho-Tyr705-STAT3; Santa Cruz Biotechnology Inc. Dallas, Texas, USA: FOXM1 (C-20), p21; GeneTex Inc. Irvine, CA, USA: GAPDH; Abcam plc Cambridge, UK: aurora A; PROGEN GmbH Heidelberg: chromogranin A (klon LK2H10)); BD Transduction Laboratories, Franklin Lakes, NJ, USA: STAT3; Sigma-Aldrich: beta-tubulin) and secondary antibodies (Dako Deutschland GmbH, Hamburg, Germany: swine anti rabbit IgG-HRP, goat anti-mouse IgG-HRP; Santa Cruz Biotechnology Inc: donkey anti-goat IgG-HRP) were applied. Antibody binding was detected with ECL™ prime Western Blotting detection reagent (Amersham™ GE healthcare) and documented by Fujifilm LAS-4000 luminescent image analyzer. For re-probing, membranes were incubated in acidic glycine buffer (0.2 M glycine, 1% SDS, 0.1% Tween 20, pH 2.2). Chemiluminescence signals were densitometrically detected with Multi Gauge V3.1. Values of three independent experiments were normalized to an internal control and statistical assessment was performed with IBM SPSS Statistics 20.
7
Affinity Purification of STAT3 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EAH-Sepharose
4B containing free amino groups with 11-atom spacer arms was used
to couple inS3-54 with the carbodiimide coupling method according
to manufacturer’s instructions. Control EAH-Sepharose was prepared
exactly the same way without inS3-54. Since inS4–54 is orange
in color, the conjugation of inS3-54 to EAH-Sepharose was verified
by monitoring the color change of the EAH beads.
For the pull-down
assay, inS3-54-conjugated and control beads equilibrated with binding
buffer (10 mM MES/NaOH, pH 6.5, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM KCl, 0.5% NP-40) were blocked with
10% milk in the binding buffer containing 0.2 mM PMSF and protease
inhibitor cocktails followed by incubation with 60 μg of lysate
of H1299 cells harboring FLAG-STAT3 or 1 μg of purified STAT3
(Sigma) at RT for 1 h. The unbound proteins were removed by washing
7 times, and the bound proteins were separated by SDS-PAGE followed
by analysis using Western blot or silver staining. For competition,
cell lysate was preincubated with 10 μM inS3-54, DMSO vehicle
or an irrelevant compound control at RT for 1 h prior to the pull-down
assay. For elution, following binding STAT3 using inS3-54-conjugated
beads as described above, the protein–bead complex were eluted
using vehicle control, 300 μM inS3-54 or the irrelevant compound
in binding buffer containing 20% DMSO.
4B containing free amino groups with 11-atom spacer arms was used
to couple inS3-54 with the carbodiimide coupling method according
to manufacturer’s instructions. Control EAH-Sepharose was prepared
exactly the same way without inS3-54. Since inS4–54 is orange
in color, the conjugation of inS3-54 to EAH-Sepharose was verified
by monitoring the color change of the EAH beads.
For the pull-down
assay, inS3-54-conjugated and control beads equilibrated with binding
buffer (10 mM MES/NaOH, pH 6.5, 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM KCl, 0.5% NP-40) were blocked with
10% milk in the binding buffer containing 0.2 mM PMSF and protease
inhibitor cocktails followed by incubation with 60 μg of lysate
of H1299 cells harboring FLAG-STAT3 or 1 μg of purified STAT3
(Sigma) at RT for 1 h. The unbound proteins were removed by washing
7 times, and the bound proteins were separated by SDS-PAGE followed
by analysis using Western blot or silver staining. For competition,
cell lysate was preincubated with 10 μM inS3-54, DMSO vehicle
or an irrelevant compound control at RT for 1 h prior to the pull-down
assay. For elution, following binding STAT3 using inS3-54-conjugated
beads as described above, the protein–bead complex were eluted
using vehicle control, 300 μM inS3-54 or the irrelevant compound
in binding buffer containing 20% DMSO.
8
Western Blot Analysis of Signaling Proteins
9
Investigating Molecular Mechanisms in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA). The Wnt-1 ELISA kit was from USCN Life Science (Houston, TX).
10
Western Blot Analysis of Signaling Proteins
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!