Resting CD4+ T cells were labeled with anti-CD4-FiTC antibody (Biolegend, #357406) and isolated from human PBMCs by magnetic activated cell sorting (MACS, Miltenyi) using anti-FiTC microbeads (Miltenyi, Cat#130-048-701) following manufacturer instructions. Then, CD4+ T cells were activated for three days with ImmunoCult Human CD3/CD28 T Cell Activator (StemCell, #10971) following manufacturer instructions in the presence of 20 ng/mL IL2 (Novartis, #709421). After activation, cells were expanded for 5 days in the presence of 20ng/mL IL2. Then, cells were starved of IL2 for 24 hours before stimulation as indicated and 105 cells were used per experiment. Cells were attached to coverslips by incubating them at 37°C for 1 hour in PBS, then PBS was replaced with RPMI supplemented with 10% FBS and cells stimulated as described. After stimulation, cells were fixed with 2% formaldehyde for 10 minutes at RT, permeabilized with ice-cold methanol for 20 minutes on ice and stained with anti-STAT3 (Cell Signaling, #9139S) and anti-CDK8 (Invitrogen, #PA1-21780) or anti-STAT3 (Cell Signaling, #9139S) and anti-CDK9 (Cell Signaling, #2316S) for Proximity Ligation Assays following manufacturer instructions (Sigma, #DUO92008).
Anti stat3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Germany, China
Anti-STAT3 is a primary antibody product from Cell Signaling Technology. It is designed to detect STAT3 (signal transducer and activator of transcription 3), a transcription factor involved in various cellular processes. This antibody can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry.
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Lab products found in correlation
Anti p stat3, by Cell Signaling Technology (146 mentions)
Anti phospho stat3, by Cell Signaling Technology (93 mentions)
Anti akt, by Cell Signaling Technology (64 mentions)
Anti gapdh, by Cell Signaling Technology (50 mentions)
Anti jak2, by Cell Signaling Technology (43 mentions)
Anti β actin, by Merck Group (40 mentions)
Anti β actin, by Cell Signaling Technology (37 mentions)
Anti stat1, by Cell Signaling Technology (35 mentions)
Anti erk1 2, by Cell Signaling Technology (33 mentions)
Anti erk, by Cell Signaling Technology (32 mentions)
Anti p akt, by Cell Signaling Technology (29 mentions)
Anti phospho stat3 tyr705, by Cell Signaling Technology (28 mentions)
Anti p erk, by Cell Signaling Technology (25 mentions)
Anti e cadherin, by Cell Signaling Technology (24 mentions)
Anti gapdh, by Santa Cruz Biotechnology (23 mentions)
Anti β actin, by Santa Cruz Biotechnology (23 mentions)
Pvdf membrane, by Merck Group (21 mentions)
Anti pstat3 tyr705, by Cell Signaling Technology (19 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Anti vimentin, by Cell Signaling Technology (18 mentions)
536 protocols using anti stat3
1
Activation and Stimulation of CD4+ T Cells
2
Immunoprecipitation of STAT Proteins
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Human CD8+ T cells were treated indicated cytokines and anti-CD3/28 crosslinking for 30 min at 37°C and then lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) and Protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 10 min at 4°C. IP was performed using anti-STAT3 (#4904S, 1:50, Cell signaling Technology) Ab or nomal rabbit IgG (#2729, Cell Signaling Technology) on a rotor for 16 h at 4°C. Protein A/G PLUS-Agarose beads (Santa-Cruz Biotechnology, Heidelberg, Germany) were added into the samples and incubated on a rotor for 4 h at 4°C. After 5 times of washing with the lysis buffer, immunoprecipitates were eluted by boiling with the SDS-loading buffer. Precipitated proteins were analyzed by SDS-PAGE, followed by transferring to PVDF membranes, immunoblotted with the indicated Abs, and developed using a chemiluminescent detection method. The Abs with the indicated dilutions were as follows: anti-STAT1 (#9172, 1:1,000), anti-STAT3 (#4904S, 1:2,000), and anti-STAT5 (#94205, 1:1,000, all from Cell Signaling Technology).
3
Protein Expression Quantification by SDS-PAGE and Immunoblotting
4
Immunofluorescence and Ki-67 Staining Protocol
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For immunofluorescence, cells in chamber slides were stained with anti-STAT3 (Cell Signaling) overnight at 4°C, following by secondary staining with the anti-rabbit Alexa Fluor 488–conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Images were taken by fluorescence microscope (Olympus Imaging America, Inc.). The immunochemical staining of Ki-67 was performed on 5-mm paraffin-embedded tumor sections as previously described (Cina et al., 1997 (link)).
5
Western Blotting Analysis of Viral and Cellular Proteins
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Expression of viral and host cellular proteins was detected by Western blotting according to the procedure described previously (10 (link)). Equal amounts (10 to 30 μg) of protein samples were separated using 10 to 12% SDS-PAGE gels and transferred to 0.2-μm nitrocellulose membranes. Anti-PSMA1 (Invitrogen; catalog no. PA1-963), anti-PSMA2 (Cell Signaling; catalog no. 2455), anti-PSMA6 (Invitrogen; catalog no. PA576058), anti-beta-actin (Cell Signaling; catalog no. 3700S), anti-STAT3 (Cell Signaling; catalog no. 9139S), anti-CST3 (Cell Signaling; catalog no. 4280), and in-house-prepared IAV mouse-anti-NS1 and mouse-anti-NP (93 (link)) were used to detect specific proteins. Appropriate secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse or anti-rabbit (Cell Signaling; catalog no. 7076 and 7074, respectively) were used to detect immune complexes. Protein bands were developed with ECL reagents and captured by Alpha Innotech FluorChemQ MultiImage III. The differences in protein expression were determined by measuring band intensities with ImageJ 1.50i (NIH, USA). The data were analyzed and graphically presented by GraphPad Prism v 9.1.0 software.
6
Gene Regulation Techniques in Cell Biology
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siRNA against WASF3 and the pLKO lentiviral vectors containing an shRNA against SHOX2 or STAT3 were purchased from Open Biosystems (Huntsville, AL). Coding sequences of human HA-tagged WASF3 and FLAG-tagged SHOX2 were respectively cloned into pCDH-CMV-MCS-EF1 lentivector (System Biosciences, Mountain View, CA) as described previously [8 (link), 13 (link)]. The pGL-WASF3 promoter constructs (-350/+494, -650/+494, -1250/+494) were generated as described previously [16 (link)]. The following primary antibodies were used in Western blot assays: anti-WASF3, anti-STAT3, anti-pSTAT3 (Tyr 705), and anti-E-cadherin antibodies (Cell Signaling Technology, MA); anti-HA, anti-FLAG, and anti-β-Actin antibodies (Sigma-Aldrich, MO); and anti-SHOX2 antibody (Abcam, MA). The STAT3 inhibitor S3I-201 was obtained from Selleckchem (Houston, TX).
7
Gastric Cancer Protein Expression Analysis
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Total protein was collected from gastric cancer cell lines and xenograft from nude mice using RIPA lysis buffer for 30 min at 4°C containing protease inhibitors, and the homogenates were centrifuged at 12,000 × g for 20 min at 4°C. Protein concentration was estimated by a BCA Protein kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of proteins (25 μg) were separated by 10–15% SDS-PAGE and transferred into nitrocellulose membrane (Millipore). After blocking with 5% fat-free milk overnight at 4°C, the blots were incubated with anti-PRAKK1 (Abcam), anti-PCNA (Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-p-ERK1 (Abcam), anti-ERK1 (Abcam), anti-p-STAT3 (Abcam), anti-STAT3 (Cell Signaling Technology), anti-p-JNK1 (Abcam), anti-JNK1 (Abcam), anti-p-Akt (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology) antibody overnight at 4°C. The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000; Beyotime) for 1 h at 37°C. The membranes were developed using an enhanced chemiluminescence (ECL) kit (Applygen Technologies, Beijing, P.R. China) following the manufacturer’s instructions.
8
Western Blot Analysis of Signaling Proteins
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Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P+ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).
9
Cell Lysis and Western Blot Analysis
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Whole-cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described24 (link). Nuclear lysates were prepared with the NE-PER Kit (Pierce) following manufacturer’s recommendation. Western blot analyses were performed with anti-MKL1 (Santa Cruz, sc-32909), anti-collagen type I (Rockland, 600-401-103), anti-α-SMA (Abcam, ab5694), anti-STAT3 (Cell Signaling Technology, 9132), anti-TWIST1 (Abcam, ab50887), anti-VE-Cadherin (Cell Signaling Technology, 2158), anti-PECAM1 (Proteintech, 11265-1), anti-α-tubulin (Sigma, T5168), anti-Lamin B (Santa Cruz, sc-6216), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.
10
Western Blot Analysis of STAT3
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Cecum samples were homogenized with a handheld homogenizer in 300µL of RIPA buffer (Thermo Fisher) supplemented with complete protease inhibitors and phosstop phosphatase inhibitors (Roche). 5 µg of protein was subject to PAGE and blotted with anti-GAPDH (Cell Signaling 2118), anti-STAT3 (Cell Signaling 4904), or anti-pSTAT3 (Y705) (Cell Signaling 9145).
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