Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma), and 1% protease inhibitors cocktail (Merck). Protein extracts were quantitated and loaded on 8% to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred onto a PVDF membrane (Millipore). The membrane was incubated with primary antibody, washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce). Detection was performed by using a chemiluminescent Western detection kit (Cell Signaling Technology). The antibodies used were Anti-caspase-3, Anti-caspase-9, Anti-PARP, Anti-pSTAT3 (Y705), Anti-Jak2, Anti-pJak2 (Y1007/Y1008), Anti-pSTAT3 (S727), Anti-STAT1, Anti-pSTAT1 (Y701), Anti-STAT5, and Anti-pSTAT5 (Y694) (Cell Signaling Technology), Anti-Lamin B, Anti-Cyclin D1 (Santa Cruz Biotechnology), Anti-Mcl-1, Anti-STAT3 (Proteintech), and Anti-GAPDH (Abmart) antibodies.
Anti p stat3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
Anti-p-STAT3 is a laboratory reagent that detects the phosphorylated form of the transcription factor STAT3. It is used to monitor the activation state of STAT3 in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti stat3, by Cell Signaling Technology (146 mentions)
Anti p akt, by Cell Signaling Technology (37 mentions)
Anti akt, by Cell Signaling Technology (34 mentions)
Anti p erk, by Cell Signaling Technology (24 mentions)
Anti gapdh, by Cell Signaling Technology (23 mentions)
Anti erk, by Cell Signaling Technology (21 mentions)
Anti p jak2, by Cell Signaling Technology (19 mentions)
Anti β actin, by Cell Signaling Technology (19 mentions)
Anti jak2, by Cell Signaling Technology (18 mentions)
Anti β actin, by Santa Cruz Biotechnology (18 mentions)
Anti p erk1 2, by Cell Signaling Technology (16 mentions)
Anti erk1 2, by Cell Signaling Technology (15 mentions)
Anti β actin, by Merck Group (15 mentions)
Anti gapdh, by Santa Cruz Biotechnology (13 mentions)
Pvdf membrane, by Merck Group (12 mentions)
Anti p stat1, by Cell Signaling Technology (11 mentions)
Anti e cadherin, by Cell Signaling Technology (10 mentions)
Anti stat3, by Santa Cruz Biotechnology (10 mentions)
Anti p p65, by Cell Signaling Technology (9 mentions)
Anti c myc, by Cell Signaling Technology (8 mentions)
249 protocols using anti p stat3
1
Western Blot Analysis of Apoptosis and Signaling
2
Immunological Profiling of Tumor Microenvironment
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Antibodies used in immunohistochemistry: Anti-CD68 (1:1000, Sigma, Cat#HPA048982, RRID : AB_2680587), anti-CD4 (1:500, Abcam, Cat#ab133616, RRID : AB_2750883), anti-CD8 (1:200, Cell Signaling Technology, Cat#85336,RRID : AB_2800052), anti-FOXP3 (1:100, Abcam, Cat#ab20034, RRID : AB_445284), anti-CD206 (1μg/ml, Abcam, Cat#ab64693, RRID : AB_1523910), Anti-TNC (1:100, Abcam, Cat#ab108930, RRID : AB_10865908).
Antibodies used in immunofluorescence: Anti-TNC (1:50, Santa Cruz Biotechnology, Cat#SC13578, RRID : AB_628341), anti-CA9 (1:100, NOVUS, Cat#NB100-417, RRID : AB_10003398), anti-Iba1 (1:100, Cell Signaling Technology, Cat#17198S, RRID : AB_2820254). Secondary Antibodies: Anti-mouse IgG (1:100, Invitrogen, Cat#2266877), anti-rabbit IgG (1:200, Invitrogen, Cat#2273718).
Antibodies used in western blotting: Anti-TNC (1:500, Santa Cruz Biotechnology, Cat#B1120),anti-CD11b (1:1000, Cell Signaling Technology, Cat#17800S), anti-p-STAT3 (1:1000, Cell Signaling Technology, Cat#9145S, RRID : AB_2491009), anti-α-Tubulin (1:5000, ABclonal, Cat#AC012, RRID : AB_2768341). Secondary Antibodies: anti-rabbit IgG (1:1000, Cell Signaling Technology, Cat#7074P2, RRID : AB_2099233), Anti-mouse IgG (1:1000, Cell Signaling Technology, Cat#7076P2, RRID:AB_330924).
Antibodies used in immunofluorescence: Anti-TNC (1:50, Santa Cruz Biotechnology, Cat#SC13578, RRID : AB_628341), anti-CA9 (1:100, NOVUS, Cat#NB100-417, RRID : AB_10003398), anti-Iba1 (1:100, Cell Signaling Technology, Cat#17198S, RRID : AB_2820254). Secondary Antibodies: Anti-mouse IgG (1:100, Invitrogen, Cat#2266877), anti-rabbit IgG (1:200, Invitrogen, Cat#2273718).
Antibodies used in western blotting: Anti-TNC (1:500, Santa Cruz Biotechnology, Cat#B1120),anti-CD11b (1:1000, Cell Signaling Technology, Cat#17800S), anti-p-STAT3 (1:1000, Cell Signaling Technology, Cat#9145S, RRID : AB_2491009), anti-α-Tubulin (1:5000, ABclonal, Cat#AC012, RRID : AB_2768341). Secondary Antibodies: anti-rabbit IgG (1:1000, Cell Signaling Technology, Cat#7074P2, RRID : AB_2099233), Anti-mouse IgG (1:1000, Cell Signaling Technology, Cat#7076P2, RRID:AB_330924).
3
Flow Cytometry Immunophenotyping Protocol
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Flow cytometry: commercial antibodies anti-CD86-FITC (clone 2231 FUN-1), CD274 (PD-L1)-FITC (clone MIH1), CD273 (PD-L2)-PE (clone MIH-18), HLA-DR-PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) were purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from Beckman Coulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2), IL-10-PE (clone JES3-9D7), KI-67-PE (clone Ki-67) were purchased from BioLegend; CD14-PE-DL594 (clone MEM-15), CD11c-APC (clone BU15), CD3-AF700 (clone MEM-57), CD8-PE-Dy590 (clone MEM-31) were purchased from Exbio; CD85k (ILT-3)-PE (clone 293623), CD85d (IL-T4)-FITC (clone 287219) were purchased from R&D Systems. For western blot, anti-p-p38 MAPK, anti-p-ERK1/2, anti-p-JNK/SAPK, anti-p-IκB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-p-p70S6K, anti-p38 MAPK, anti-ERK1/2, anti-JNK/SAPK and anti-STAT3 Ab were purchased from Cell Signaling Technology; anti-actin was from BioLegend.
4
Immunofluorescence Staining of Spinal Cord
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Tissue processing and immunofluorescence was performed as described by Muñoz et al. (2015 ) with minor modifications. Briefly, spinal cord cryosections were permeabilized with 100% methanol at −20°C for 10 min and then with phosphate‐buffered saline (PBS) + 0.2% Triton X‐100 (PBST) at room temperature for 10 min, blocked in PBST + 10% goat serum, incubated with primary antibodies overnight at 4°C, secondary antibodies for 2 hr at room temperature and stained with TOTO‐3 or Hoechst 33342. Antibodies used were rabbit mAb anti‐pSTAT3 (1:200, Cell Signaling Technology, D3A7), mouse mAb anti‐Sox2 (1:200, Cell Signaling Technology, L1D6A2), mouse mAb anti‐Islet1/2 (1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; 39.4D5), mouse mAb anti‐CD45 (1:50; Xenopus laevis Resource for Immunobiology, Rochester, NY, USA; CL21) and AlexaFluor 488 or 555 (1:500; Invitrogen, Carlsbad, CA, USA) as secondary antibodies.
Samples were photographed using an inverted fluorescence microscope (Microfluo Olympus IX71) or a confocal microscope (FV‐1000 Olympus Confocal Laser Scanning Microscope). Total cell counts for quantification analysis were determined using the ImageJ cell counter plugin.
Samples were photographed using an inverted fluorescence microscope (Microfluo Olympus IX71) or a confocal microscope (FV‐1000 Olympus Confocal Laser Scanning Microscope). Total cell counts for quantification analysis were determined using the ImageJ cell counter plugin.
5
ChIP-qPCR Analysis of C/EBPα and p-STAT3
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Cells were fixed with 1% formaldehyde for 10 minutes at 37°C. The crude nuclei were subjected to sonication to produce chromatin fragments of about 500 bp length. The antibodies included anti-C/EBPα (catalog PA5-77911, Thermo Fisher Scientific) and anti–p-STAT3 (catalog 9145, Cell Signaling Technology). Isotype IgG (catalog 61656, Cell Signaling Technology) was used as a negative control. Primary antibodies (2–5 μg) and samples were incubated overnight at 4°C with gentle shaking. Primer sequences for ChIP-qPCR are listed in Supplemental Table 2 . ChIP regions within the CCL3 and C/EBPα promoter are displayed in Supplemental Figures 5 and 7 .
6
Comprehensive Protein Expression Analysis
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Western blot analysis was conducted as previously described [24 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, and anti-c-Met (Epitomics, Burlingame, CA, USA), anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin, anti-c-myc, anti-EGFR, anti-p-EGFR (Tyr1068), anti-GSK3β, anti-p-GSK3β (Ser9), anti-AKT (pan), anti-p-AKT (Ser473), anti-stat3, anti-p-stat3 (Tyr705) and anti-CREB1 (Cell Signaling Technology, Beverly, MA).
7
Western Blot Analysis of Protein Markers
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For the westernblot assay, cells were harvested in ice-cold PBS 48 h after transfection and lysed on ice in cold-modified radioimmunoprecipitation buffer supplemented with protease inhibitors. Protein concentration was determined using the BCA Protein Assay Kit (Bio-Rad, CA, USA) and equal amounts of protein were analyzed by SDS-PAGE. Gels were electroblotted onto nitrocellulose membranes (Millipore, WI, USA). After blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 2 h, membranes were incubated at 4°C over night with primary antibody. Primary antibodies used were anti-STAT3, anti-p-STAT3, anti-MMP2, anti-BCL2, anti-E-cadherin, anti-KIRT1, anti-SNAI2, anti-N-cadherin (Cell Signaling, USA) and GAPDH (Zhong-Shan JinQiao, China). Then, membranes were incubated with respective second antibodies and detected by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system (ECL) (Millipore, WI, USA). The experiment was repeated three times.
8
Western Blot Analysis of Protein Markers
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HTMCs were washed twice in cold PBS. Total protein was extracted using RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS,1.0% Triton X-100, 5 mM EDTA, pH 8.0) with 10 × protease inhibitor cocktail (Roche, San Francisco, CA, USA). Total protein extracts (20-100 μg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membrane. Membranes were blocked with 5% nonfat dry milk and incubated overnight with the primary antibodies, antifibronectin, anticollagen I (Abcam, Boston, MA, USA), anti-EGFR, anti-pSTAT3, anti-STAT3, anti-pAKT, anti-AKT, anti-pERK, anti-ERK (Cell Signaling Technology, Boston, MA, USA), and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. After being incubated with secondary antibodies, the antibody-antigen complexes were detected using the Chemiluminescent HRP Substrates (Millipore, Billerica, MA, USA).
9
Evaluating EGFR Signaling Dynamics
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Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).
10
EMT Regulation and Src/STAT3 Signaling
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The study protocol was performed according to the principles of the Declaration of Helsinki and the ethics committee of the Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu Province, China. Informed consent was obtained from all patients.
The following primary antibodies were purchased from Abcam (Cambridge, England): anti-E-cad, anti-Vim, anti-Snail, anti-N-cad, anti-Twist, anti-PCNA, anti-Bax, anti-Bcl-2, anti-ZEB1, anti-NEDD9, anti-Myb, and anti-GAPDH. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-t-SRC, anti-p-SRC, anti-t-STAT3, and anti-p-STAT3. PP1, an SRC inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).
The following primary antibodies were purchased from Abcam (Cambridge, England): anti-E-cad, anti-Vim, anti-Snail, anti-N-cad, anti-Twist, anti-PCNA, anti-Bax, anti-Bcl-2, anti-ZEB1, anti-NEDD9, anti-Myb, and anti-GAPDH. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-t-SRC, anti-p-SRC, anti-t-STAT3, and anti-p-STAT3. PP1, an SRC inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).
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