DN T cells purified from MRL/lpr mice administered NCTD or control, as well as DN T cells treated with or without NCTD were homogenized in RIPA buffer (Beyotime Biotechnology) supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology). Cell lysates (40 μg) were separated on SDS-PAGE and immunoblotted using antibodies against the following proteins: phospho-STAT3 (Y705) (Cell Signaling Technology, Danvers, MA, USA), STAT3 (Cell Signaling Technology), phospho-AKT (Thr308) (Cell Signaling Technology), AKT (Cell Signaling Technology) and β-actin (Sigma-Aldrich). CD4+ T cells treated with or without NCTD (10 μg/mL) were stimulated with IL-6 or TGF-β1 (Peprotech) in time gradient and immunoblotted using antibodies against the following proteins: phospho-STAT3 (Y705) (Cell Signaling Technology), STAT3 (Cell Signaling Technology), p-Smad2 (Cell Signaling Technology), p-Smad3 (Abcam Ltd), Smad2 (Cell Signaling Technology), Smad3 (Abcam Ltd) and β-actin (Sigma-Aldrich).
Phospho stat3 y705
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-STAT3 (Y705) is a lab equipment product that detects the phosphorylation of STAT3 at tyrosine 705. This modification is a key regulatory event in STAT3 activation and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (23 mentions)
β actin, by Merck Group (9 mentions)
Phospho akt s473, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Total stat3, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Stat1, by Cell Signaling Technology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Phospho stat3 s727, by Cell Signaling Technology (3 mentions)
Phospho stat1 y701, by Cell Signaling Technology (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Stat3, by BD (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
E cadherin, by BD (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Phospho histone h2ax s139, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
P smad3, by Abcam (2 mentions)
Smad2, by Cell Signaling Technology (2 mentions)
50 protocols using phospho stat3 y705
1
NCTD Modulates STAT3 and AKT Signaling
2
Investigating STAT3 and Smad Signaling in DN T Cells and CD4+ T Cells
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DN T cells purified from MRL/lpr mice administrated with NCTD or control, as well as DN T cells treated with or without NCTD were homogenized in RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology). Cell lysates (40 μg) were separated on SDS-PAGE and immunoblotted using antibodies against the following proteins: phospho-STAT3 (Y705) (Cell Signaling Technology, MA, USA), STAT3 (Cell Signaling Technology) and β-actin (Sigma-Aldrich). CD4+ T Cells treated with or without NCTD (10 μg/ml) were stimulated with IL-6 or TGF-β1 in time gradient and immunoblotted using antibodies against the following proteins: phospho-STAT3 (Y705) (Cell Signaling Technology), STAT3 (Cell Signaling Technology), p-Smad2 (Cell Signaling Technology), p-Smad3 (Abcam Ltd), Smad2 (Cell Signaling Technology), Smad3 (Abcam Ltd) and β-actin (Sigma-Aldrich).
3
Antibody Characterization Protocol
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We used the following primary antibodies for the proteins listed: GRP78 from Dr. Parkash S. Gill at USC (MAb159), CD44v3 from Thermo Fisher Scientific (BMS144, Waltham, MA), glutathione S-transferase (GST) tag from Santa Cruz Biotechnology, Inc. (sc-138, Dallas, TX), HA tag from Santa Cruz Biotechnology, Inc. (sc-805, Dallas, TX), FLAG M2 from Sigma-Aldrich (F1804, St. Louis, MO), Annexin II from BD Biosciences (610068, San Jose, CA), β-actin from Sigma-Aldrich (A5316, St. Louis, MO), Phospho-STAT3(Y705) from Cell Signaling Technology (9145, Danvers, MA), STAT3 from BD Biosciences (610190, San Jose, CA), Cyclin D1 from Santa Cruz Biotechnology, Inc. (sc-753, Dallas, TX), cleaved Caspase-3 from Cell Signaling Technology (9661, Danvers, MA), and cleaved PARP from Cell Signaling Technology (5625, Danvers, MA). Secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX), LI-COR Biosciences (Lincoln, NE), and Enzo Life Sciences (Farmingdale, NY).
4
Comprehensive Protein Analysis by Western Blot
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Total protein was resolved by SDS-PAGE (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences) and probed with antibodies specific for Phospho-STAT3 (S727) (ab32143, abcam), Phospho-STAT3 (Y705) (9131, Cell Signalling Technology (CST)), Total STAT3 (C-20: sc-482, Santa Cruz Biotechnology), Phospho-STAT5 (Y694) (9314, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), involucrin (SY5, Santa Cruz Biotechnology), HPV18 E6 (G-7, Santa Cruz Biotechnology), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (C1P5, Santa Cruz Biotechnology), HPV 16 E7 (ED17, Santa Cruz Biotechnology), Phospho-ERK1/2 (Thr202/Tyr204) (43705, CST), Phospho-JNK (Thr183/Tyr185) 4668, CST), Phospho-p38 (Thr180/Tyr182) (9211, CST), Bcl xL (H-62, Santa Cruz Biotechnology), Cyclin D1 (A-12, Santa Cruz Biotechnology) p53 (FL-393, Santa Cruz Biotechnology), p21 (2947, CST), FLAG (F3165, Sigma), GFP (B-2: sc-9996, Santa Cruz Biotechnology) and GAPDH (G-9, Santa Cruz Biotechnology). Western blots were visualized with species-specific HRP conjugated secondary antibodies (Sigma) and ECL (Thermo/Pierce).
5
Oxidative Stress-Induced Signaling Pathways
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TauCl as a crystalline sodium salt (MW 181.57) was prepared as described previously [12 (link)]. Primary antibody for protein modified with 4-hydroxynonenal (4-HNE) was purchased from Japan Institute for the Control of Aging (JaICA), Nikken SEIL Co., Ltd. (Shizuoka, Japan). Primary antibodies for cyclooxygenase-2 (COX-2), Signal transducer and activator of transcription (STAT3), phospho-STAT3Y705, cyclin D1, α-tubulin, Kelch-like ECH-associated protein 1 (Keap1) and lamin B1 were provided by Cell Signaling Technology (Danvers, MA, USA), and those for Nrf2 and heme oxygenase-1 (HO-1) were supplied from Abcam (Cambridge, MA, USA) and Enzo Life Sciences (Farmingdale, NY, USA), respectively. Nuclear factor-κB (NFκB) p65 and phospho-NFκB p65 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for caspase-3 and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Western Blot Analysis of Stem Cell Regulators
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Cells and tumors were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, and Halt protease inhibitor cocktail [Pierce]) and total protein was quantified using Bio-Rad protein assay reagent. An equal amount of protein was loaded on 10% or 15% SDS-PAGE gels and immunoblotted. STRA6 antibody was generated in the Lerner Research Institute Molecular Biotechnology core as previously described (Berry et al., 2013 (link), Bouillet et al., 1997 (link)). RBP4 antibody was purchased from Atlas Antibodies (HPA001641) and serum RBP4 was blotted using antibody from Dako (#A0040). Antibodies to NANOG (#4903), SOX2 (#3579), STAT3 (#4904), and phospho-STAT3 (Y705) (#9145) were from Cell Signaling Technology. RARβ (SC-552), βActin (SC-47778), and GAPDH (SC-32233) antibodies were from Santa Cruz Biotechnology.
7
Western Blot Analysis of Protein Expression
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Cells were lysed in ice-cold RIPA cell buffer (P0013B, Beyotime Biotechnology, China) supplemented with protease inhibitor cocktail. The proteins (40–100 μg) were separated on 10–15% PAGE gels and electrotransferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membrane was incubated with specific primary antibody and horseradish peroxidase-conjugated secondary antibody. The antibodies used are Flag (GNI4110-FG-S, GNI, Japan, 1:1000), HA (GNI4110-HA-S, GNI, Japan, 1:1000), Stat3 (12640S, Cell Signaling Technology, USA,1:1000), phospho-Stat3Y705 (9131S, Cell Signaling Technology, USA, 1:1000), Socs3 (sc-73045, Santa Cruz, 1:1000), Prdm14(D221722, BBI, China, 1:1000), H3K27me3 (39055, Active Motif, China, 1:1000), H3 (4620s,Cell Signaling Technology, USA, 1:1000) and β-Tubulin (200608, ZENBIO, China, 1:2000). The band density was analyzed with ImageJ according to ImageJ User Guide. Briefly, we inverted the greyscale images and select sample bands with rectangular selections. The proteins levels were normalized with respect to the β-Tubulin level, and the grayscale ratio of protein/β-Tubulin was calculated and visualized with GraphPad Prism 8.0.
8
Protein Expression and Signaling Assay
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Cell lysates were prepared in RIPA buffer containing Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail (Pierce, Rockford, IL, USA), and were centrifuged at 3500 r.p.m. for 10 min at 4 °C. Protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins (10–15 μg) from each sample were subjected to sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. Target proteins were detected using specific antibodies against phospho-STAT3 (Y705) (cat# 9145S), phospho-JAK2 (Y1007/Y1008) (cat# 3771), STAT3 (cat# 12640), JAK2 (cat# 3230 S), and β-actin (cat#3700) (purchased from Cell Signaling Technology, Beverly, MA). Antibodies against phospho-GP130 (Ser782) (cat#sc-377572) and GP130 (cat# sc-376280) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For nuclear and cytoplasmic protein fractions, we used NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology Inc.) following the manufacturer’s instructions. The cytoplasmic and nuclear protein fractions were normalized to Anti-NaKATPase (cat# ab76020) (Abcam, Cambridge, MA) and lamin B (cat# sc-374015) (Santa Cruz, CA), respectively. All antibodies were used at 1:1000 dillution.
9
Antibody Detection Techniques in Cell Signaling
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Antibodies used in this study that were purchased from Cell Signalling Technologies (Danvers, MA, USA) include those that detect: Phospho-Akt/non-phospho Akt (#4060/#2920), phospho-p38/non-phospho p38 (#9211/#9212), phospho-Erk/non-phospho Erk (#4377/#4696), phospho-JNK/JNK (#9251/#9252), phospho-JAK1 (#3331), JAK2 (#3230), phospho-STAT1 (#7649), phospho-STAT3 Y705 (#9145), phospho-STAT6 (#9361), phospho-IκBα/non-phospho-IκBα (#9241/#9242), phospho-p65/non-phospho p65 (#3033/#8242), iNOS (#13120) and IL-1b (#12242). Antibodies from Santa-Cruz Biotechnology (Santa Cruz, CA, USA) include those targeting HO-1 (sc-136960), IRF4 (sc-28696), α-tubulin (sc-5286), IRF3 (sc-33641). Anti-HIF-1α antibody (#A300-286A) was from Bethyl laboratories, (Montgomery, TX, USA), E-Cadherin (#610181) was purchased from BD Biosciences, (Wokingham, England, UK). Mouse and rabbit anti-PDLIM2 antibodies have been described previously (14 (link)).
10
OSM Signaling Pathway Activation in T47D Cells
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T47D cells were plated on 24-well plates at 70–75% confluency and allowed to adhere overnight. Cells were serum starved with serum-free RPMI media for 4 h and subsequently treated with either commercially available recombinant human OSM (chOSM) (PeproTech, Rocky Hill, NJ; Cat# 300-10 T) or rhOSM at specified concentrations for 30 min. Afterwards, cell lysates were collected with 1X Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA; Cat# 7018). Lysates were run on an SDS-PAGE gel and transferred onto a nitrocellulose membrane via semi-dry transfer. Blots were rinsed in ddH2O and allowed to dry completely before being blocked with LiCor Odyssey PBS blocking buffer (LiCor, Lincoln, NE; Cat# 927-4000) for 1 h. After blocking, primary antibodies (1:1000) suspended in blocking buffer were applied to the membrane, shaken for 1 h at room temperature, and incubated overnight at 4 °C. Membranes were then washed 6 × 5 min with 1X PBS supplemented with 0.5% Tween and secondary antibodies (1:15,000) suspended in blocking buffer were applied for 45 min. A final wash step of 6 × 5 min with PBS-T, membranes were imaged at 700 nm using the LiCor Odyssey CLx imaging system. Antibodies : phospho-STAT3 (Y705) (Cell Signaling Technology, Danvers, MA; Cat# 9145), beta-Actin (Cell Signaling Technologies; Cat# 3700), donkey anti-rabbit IRDye 800CW (LiCor, Lincoln, NE; Cat# 925-32213).
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