Total proteins from human cardiac fibroblast were separated by SDS-PAGED on 10% polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). Membranes were probed with primary antibodies for RCN-1 (Abcam; dilution 1:500), RCN-2 (Abcam; dilution 1:500), RCN-3 (Abcam; dilution 1:500), collagen I (Sigma; dilution 1:500), collagen III (Santa Cruz; dilution 1:500), Fibronectin (Millipore; dilution 1:1000), Gal-3 (ThermoFisher; dilution 1:1000), CT-1 (Abcam; dilution 1:500), connective tissue growth factor (CTGF; Torrey Pines Biolabs Inc., dilution 1/1000), ERK1/2 and ERK1/2-P (Thr202/Tyr204) at 1/1000 (Cell Signaling), Akt and Akt-P (Ser473) at 1/1500 (Cell Signaling), Stat3 and Stat3-P (Tyr705) at 1/1500 (Cell Signaling). Western blots were performed with stain-free gels for loading control. After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). After densitometric analyses, optical density values were expressed as arbitrary units. Results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. All Western Blots were performed at least in triplicate for each experimental condition.
P stat3 tyr705
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
P-STAT3 (Tyr705) is a primary antibody that detects STAT3 phosphorylated at tyrosine 705. STAT3 is a transcription factor that plays a role in cellular processes such as cell growth and survival. Phosphorylation at tyrosine 705 is important for STAT3 activation and dimerization.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (54 mentions)
β actin, by Cell Signaling Technology (16 mentions)
P stat3 ser727, by Cell Signaling Technology (16 mentions)
Gapdh, by Cell Signaling Technology (14 mentions)
β actin, by Merck Group (13 mentions)
Bcl 2, by Cell Signaling Technology (11 mentions)
P akt ser473, by Cell Signaling Technology (10 mentions)
β actin, by Santa Cruz Biotechnology (10 mentions)
P akt, by Cell Signaling Technology (8 mentions)
Bcl xl, by Cell Signaling Technology (8 mentions)
P erk, by Cell Signaling Technology (8 mentions)
Bcl xl, by Santa Cruz Biotechnology (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Gapdh, by Santa Cruz Biotechnology (7 mentions)
Mcl 1, by Cell Signaling Technology (7 mentions)
P erk1 2 thr202 tyr204, by Cell Signaling Technology (7 mentions)
Erk1 2, by Cell Signaling Technology (7 mentions)
Cyclin d1, by Cell Signaling Technology (7 mentions)
C myc, by Cell Signaling Technology (6 mentions)
P jak2, by Cell Signaling Technology (6 mentions)
157 protocols using p stat3 tyr705
1
Cardiac Fibroblast Protein Analysis
2
Stem Cell Marker Protein Detection
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Cultured cells were lysed in strong RIPA buffer containing Halt Protease Inhibitor Cocktails (Thermo, Waltham, MA). The obtained protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL). Primary antibodies targeting OCT4 (Santa Cruz Biotechnology, Santa Cruz, CA), NANOG (Abcam, Cambridge, U.K), SOX2 (Abcam), STAT3 (Cell Signaling Technology, Boston, MA), STAT3 (pTyr705) (Cell Signaling Technology) and GAPDH (Santa Cruz Biotechnology) were incubated with the proteins overnight at 4℃, followed by incubation with the appropriate HRP (horseradish peroxidase) conjugated secondary antibodies. Detection of HRP was performed using the Super Signal West Pico Chemiluminescent Substrate (Pierce).
3
Comprehensive Protein Expression Analysis
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Antibodies (Ab) against Chk1, Chk1-pSer296, Chk1-pSer317, Chk1-pSer345, p21, beta-Actin, H2AX, H2AX-pSer139, total PARP, cleaved-PARP, total caspase-3, cleaved-caspase-3, c-Myc, Bcl-2, Mcl-1, STAT3-pSer727, and STAT3-pTyr705 were purchased from Cell Signaling Technology. Anti-NFκB Ab was from Santa Cruz Biotechnology. Anti-p53 Ab (DO-1) was a gift from Dr. Borivoj Vojtesek (Masaryk Memorial Cancer Institute, Brno). Anti-rabbit and anti-mouse secondary antibodies were purchased from DakoCytomation. Anti-Ki-67-PE Ab and appropriate isotype control were purchased from BioLegend.
4
Western Blot Analysis of Cardiac Proteins
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Equal protein amounts from isolated cardiomyocytes, rat heart, and isolated mitochondria were resolved by 8-12% SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis, as previously described [26 (link)]. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline- (TBS-) Tween and were incubated with primary antibodies overnight at 4°C at the following dilutions: AMPK, phosphorylated AMPK (Thr172), STAT3, phosphorylated STAT3 at Tyr705 (p-STAT3 Tyr705), Bax, Bcl2, caspase-3, and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) 1 : 1000; phosphorylated STAT3 at Ser727 (p-STAT3 Ser727) (Cell Signaling Technology, Beverly, MA) 1 : 500. After washing with TBS-Tween, immunoreactive bands were visualized by an enzymatic chemiluminescence method and quantified with Quantity One image software.
5
Western Blot Analysis of PIAS3 and STAT3
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After transfection for 24 hours, cells were lyzed in lysis buffer (Beyotime, Shanghai, China) with the protease inhibitor (Roche, Mannheim, Germany). The protein concentration was examined with a BCA Protein Assay kit (Pierce, Rockford, IL). For sodium dodecyl sulphate polyacrylamide (SDS) gel electrophoresis, equal quantities of total protein (30 μg) were separated on 10% SDS‐PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking in nonfat milk at room temperature for 1 hour, the membranes were incubated with the specific antibodies against PIAS3 (Abcam, Cambridge, MA), STAT3 (Cell Signalling Technology, Beverly, MA), p‐STAT3 (Tyr705) (Cell Signalling Technology) and β‐actin (Cell Signalling Technology) at 4°C overnight. After washing with Tris‐buffered saline‐tween (TBS‐T), the membranes were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody at room temperature for 1‐2 hours. Protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). The Western blots were quantified using Gel‐Pro analyzer software (v4.5, Media Cybernetics, Rockville, Maryland, USA).
6
Metformin, Celecoxib, and PGE2 Effects
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T24 or RT4 cells were seeded in 6-well plates 2×105 cells/well, treated with corresponding reagents, including metformin (0, 1 mM, 5 mM, 10 mM, 20 mM), Celecoxib (20 μM YUANYE BioTECH, Shanghai, China) and PGE2(10 μM, Sigma). Cell lysates were separated on SDS-PAGE followed by western blotting assay as described [26 (link)] with the following primary antibodies: Bcl-2 (1:1000 Epitomics), Cyclin D1 (1:1000 Fantibody, China), CK14 (1:400 Abcam), STAT3 (1:2000 Abcam), OCT3/4 (1:400 Santa Cruz Biotech-nology), COX2 (1:2000 Origene), p-STAT3 (Tyr705) (1:2000 Cell Signaling), AMPK (1:700 Proteintech), p-AMPK (1:1000 Cell Signaling) and β-actin (1:5000 Cell Signaling).
7
Western Blotting of Key Signaling Proteins
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Western blotting was performed as previously described.39 (link) Briefly, total cell lysates were generated and proteins were separated on a 10% SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes. The membranes were washed and blocked. The primary antibodies and dilutions used were as follows: p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-STAT3 (Tyr705), and STAT3 (1:1000, Cell Signaling Technology, Beverly, MA, USA); TET1 (1:1000, clone ab191698, Abcam, Cambridge, MA, USA); TET2 (1:800, clone ab94580, Abcam); TET3 (1:1000, clone ab139805, Abcam); SOCS1(1:800, clone ab83493, Abcam); PTEN (1:800, clone ab31392, abcam); MMP9 (1:1000, clone ab38898, Abcam); tubulin (1:1000; clone 2A9, Abnova, Taipei, Taiwan); and GAPDH (1:5000; Millipore, Bedford, MA, USA). Samples were incubated with primary antibodies then with horseradish peroxidase- conjugated secondary antibodies. Antibody binding was detected by enhanced chemiluminescence assays.
8
Molecular Mechanisms of JAK/STAT Signaling
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The following antibodies were used: anti-CEP55 (#23891-1-AP), MMP2 (#66366-1-Ig), MMP9 (#10375-2-AP), STAT3 (#60199-1-Ig), JAK1 (#66466-1-Ig), JAK2 (#17670-1-AP), and Actin (#60008-1-Ig) (Proteintech Group, Chicago, IL, USA); and p-JAK1 Tyr1034/1035 (#74129), p-JAK2 Tyr1007/1008 (#3771), and p-STAT3 Tyr705 (#9145) (Cell Signaling Technology, Beverly, MA, USA). The biological reagent IL-6 was acquired from Sigma Chemical (St. Louis, MO, USA). JAK2 inhibitor XL019 (#S7036), STAT3 inhibitor C188-9 (#S8605) was from Selleck (Shanghai, China).
9
Protein Expression Profiling Protocol
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We obtained CB2 antibody (1:500) from Abcam Inc. (Cambridge, MA). We purchased CB1 (1:500), total STAT1 (1:1000), total STAT3 (1:1000), iNOS, NOX3, and TRPV1 (1:300) from Santa Cruz Biotechnology (Santa Cruz, CA). We bought p-STAT1Ser 727 (1:500), p-STAT3Tyr 705 (1:500), p-JAK2 (1:1000), from Cell Signaling Technology Inc. (Danvers, MA). We purchased secondary antibodies; goat anti-rabbit, donkey anti-goat and goat anti-mouse from Life Technology (Eugene, OR), and fluorescent tagged (dylight 488 and TRITC) secondary antibodies (1:500) from Jackson Immuno Laboratories (West Grove, PA).
10
Protein Extraction and Western Blot Analysis
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Total protein lysate from cells or tissues was prepared using standard RIPA lysis buffer (Sigma Chemicals, St. Louis, MO, USA). To minimize protein dephosphorylation, phosphatase-inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added into the lysis buffer. Protein concentration was then measured using a bicinchoninic acid assay (Thermo Scientific, Bonn, Germany). Fifty to 80 μg of each protein lysate was separated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were then blocked in 5% skim milk solution in TBST for 1 h at room temperature and then incubated with primary antibodies raised against PTPRD (1:500; LifeSpan BioSciences), ALDH1 (1:300; Proteintech Group, Chicago, USA), STAT3 antibody (1:500; Proteintech Group), pSTAT3 (Tyr705) (1:500; Cell Signaling Technology, Inc., Danvers, MA, USA), OCT-4 (1:300; Proteintech Group), E-cadherin (1:500; Proteintech Group), and vimentin (1:500; Proteintech Group) at 4°C overnight, and subsequently with an IRDye 800 CW-labeled secondary antibody (1:5,000). Protein bands were quantified by optical density analysis and normalized to GAPDH.
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