Immunofluorescence and immunohistochemistry were performed on formalin fixed, paraffin-embedded tumor sections as described previously (28 (link)). The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100). Immunoblotting was performed using standard methods on uncultured, macrodissected tumor protein lysates or lysates from cultured cell lines. Membranes were probed with NFATc2 (Abcam), p-NFATc2 (S213/217/221) (Abcam), Flag (Sigma-Aldrich), HA, STAT3, GSK-3β, CDK 6, p-STAT3 (Y705), p-GS (S641/645), GS (all Cell Signaling), V5 (Invitrogen) and β-actin (Sigma-Aldrich) antibodies. Densitometric analysis was carried out with ImageQuant 5.1.
Stat3
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany, Japan, Morocco
STAT3 is a protein that plays a key role in cell signaling pathways. It functions as a transcription factor, regulating the expression of genes involved in various cellular processes such as cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
P stat3, by Cell Signaling Technology (403 mentions)
Phospho stat3, by Cell Signaling Technology (152 mentions)
β actin, by Cell Signaling Technology (120 mentions)
P akt, by Cell Signaling Technology (118 mentions)
Gapdh, by Cell Signaling Technology (112 mentions)
Erk1 2, by Cell Signaling Technology (92 mentions)
β actin, by Merck Group (81 mentions)
Stat1, by Cell Signaling Technology (72 mentions)
P erk1 2, by Cell Signaling Technology (65 mentions)
β actin, by Santa Cruz Biotechnology (63 mentions)
E cadherin, by Cell Signaling Technology (62 mentions)
Bcl 2, by Cell Signaling Technology (60 mentions)
P erk, by Cell Signaling Technology (57 mentions)
Cleaved caspase 3, by Cell Signaling Technology (54 mentions)
P stat3 tyr705, by Cell Signaling Technology (54 mentions)
Gapdh, by Santa Cruz Biotechnology (52 mentions)
Vimentin, by Cell Signaling Technology (46 mentions)
P jak2, by Cell Signaling Technology (45 mentions)
P stat3 y705, by Cell Signaling Technology (45 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (39 mentions)
1 075 protocols using stat3
1
Immunofluorescence and Immunohistochemistry of Tumor Samples
2
Western Blot Analysis of Phospho-STAT3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One hundred micrograms of denatured total proteins from liver, extracted using RIPA buffer (Merck, Germany) were separated by SDS-PAGE electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, MA, USA). Membranes were overnight incubated at 4°C with 1:1000 dilution of phospho-STAT3 (Tyr705, #9138 Cell Signaling, Beverly, MA, USA), STAT3 (#4904 Cell Signaling, Beverly, MA, USA) and β-actin (#4967 Cell Signaling, Beverly, MA, USA) primary antibodies followed by 1 h incubation at RT with horse anti-mouse IgG-HRP at 1:5000 (#7076 Cell Signaling, Beverly, MA, USA) for phospho-STAT3 antibody binding and goat anti-rabbit IgG-HRP at 1:5000 (#7074 Cell Signaling, Beverly, MA, USA) for STAT3 and β-actin antibodies binding. Chemiluminescence signal was enhanced by adding ECL (SuperSignalTM West Dura Extended Duration Substrate, Thermo Fisher Scientific, MA, USA) and quantified using ImageJ 1.8 software.
3
Multiparametric Immunostaining and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies used in immunostaining were as follows: RNF20 (21615-1-AP; Proteintech, Rosemont, IL, USA), NESTIN (MAB353; Millipore, Darmstadt, Germany), ACSBG1 (ab118154; Abcam, Cambridge, UK), GFAP (G6171; Sigma, St. Louis, MO, USA), GFAP (Z0334 Dako, Santa Clara, CA, USA), GFAP (60190-1-Ig; Proteintech), GLAST (BMP009; MBL, Beijing, China), Tenascin C (110-68136; NB, Littleton, CO, USA), Ki67 (ab137876; Abcam), FLAG (F1804; Sigma), STAT3 (#9139; Cell Signaling Technology, Beverly, MA, USA). The primary antibodies used in western blotting were as follows: RNF20 (21615-1-AP; Proteintech), GLAST (20785-1-AP; Proteintech), ACSBG1 (ab118154; Abcam), GFAP (G6171; Sigma), STAT3 (#9139; Cell Signaling Technology), phospho-STAT3 (#9145; Cell Signaling Technology), Ubiquityl-Histone H2B (Lys 120) (#5546; Cell Signaling Technology), H4K16ac (07-329; Millipore), H3K9me3 (07-442; Millipore), Trimethyl-Histone H3 (Lys 27) (07-449; Millipore), H2B (ab1790; Abcam), FLAG (F1804; Sigma), HA (M20003; Abmart, Shanghai, China), β-ACTIN (20536-1-AP; Proteintech). Alexa-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used for immunostaining. IR Dye (LI-COR Biosciences, Lincoln, Nebraska, USA) 800 CW donkey anti-rabbit and 680 CW donkey anti-mouse secondary antibodies were used in western blotting.
4
Western Blot Analysis of IL-18 and Stat3 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Colon tissue samples were acquired and lyzed with RIPA buffer (Beyotime Institute of Biotechnology). Protein was quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 100 µg protein per sample was boiled for 5 min prior to separation by 10% SDS-PAGE, and then transferred onto nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk at 37°C for 1 h and incubated with corresponding primary antibodies: IL-18 (cat. no. BS6823; 1:2,000; Bioworld Technology, Inc. St. Louis Park, MN, USA), phosphorylated (p-) signal transducer and activator of transcription (Stat) 3 (cat. no. 9145; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), total Stat3 (cat. no. 12640; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. AP0063; 1:5,000; Bioworld Technology, Inc.) at 4°C overnight. Following 3 washes for 10 min in TBST, the membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. Enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) was used to develop the blots and protein was analyzed using Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
5
NCTD Modulates STAT3 and AKT Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
6
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed with 1 × lysis buffer (Cell Signaling Technology). Cell lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with antibodies against LAMA4 (Santa Cruz Biotechnology, Cat # sc-130541, RRID : AB_2296778), GAPDH (Thermo Fisher Scientific, Cat # AM4300, RRID: AB_2536381), EGFR (Cell Signaling Technology Cat# 4267, RRID : AB_2246311), ITGA6 (Santa Cruz Biotechnology Cat# sc-374057, RRID : AB_10917002), β-actin (Santa Cruz Biotechnology, Cat # sc-69879, RRID: AB_1119529), phosphorylated ERK1/2 (Cell Signaling Technology Cat# 9101, RRID : AB_331646), STAT1 (Cell Signaling Technology Cat# 9167, RRID : AB_561284), STAT3 (Cell Signaling Technology Cat# 9145, RRID : AB_2491009), and Akt (Cell Signaling Technology Cat# 9271, RRID : AB_329825), non-phosphorylated ERK1/2 (Cell Signaling Technology Cat# 4695, RRID : AB_390779), STAT1 (Cell Signaling Technology Cat# 14994, RRID : AB_2737027), STAT3 (Cell Signaling Technology Cat# 9132, RRID : AB_331588) and Akt (Cell Signaling Technology Cat# 9272, RRID : AB_329827).
7
Investigating STAT3 and Smad Signaling in DN T Cells and CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
8
Recombinant Human IL-26 Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant human IL-26 protein (MBS1362134) was purchased from MyBiosource (San Diego, CA, USA). Palmitate (P0500), metformin (D150959), methylthiazolyldiphenyl-tetrazolium bromide (MTT) (M5655), and bovine serum albumin (BSA) (A7030) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Antibodies against cyclooxygenase-2 (COX-2; 1:1000; 35-8200) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), while those against collagen type II (COL-II; 1:1000; ab34712) and matrix metalloproteinase-1 (MMP-1) (1:1000; ab134184) were purchased from Abcam (Cambridge, MA, USA), and those against IL-6 (1:1000; #12153), Erk1/2 (1:1000; #4695), phospho-Erk1/2 (1:1000; #9101), phospho-c-Jun (1:1000, #3270), phospho-p38 (1:1000, #9211), phospho-JNK (1:1000, #9251), STAT1 (1:1000, #14994), phospho-STAT1 (1:1000, #9131), STAT3 (1:1000, #9139), phospho-STAT3 (1:1000, #9131), histone H3 (1:2000, #9715), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; #5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
9
Comprehensive Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described previously (9 (link)). Samples (20 μg) were run on a 10% SDS-PAGE gel followed by blotting to a nitrocellulose membrane. Membranes were blocked and incubated with the following antibodies: TRPV4 (ACC-034, Alomone Labs), SMAD3 (9523, Cell Signaling Technology), p-SMAD 3 (9520, Cell Signaling Technology), ERK (4695, Cell Signaling Technology), p-ERK (4370, Cell Signaling Technology), JNK (9252s, Cell Signaling Technology), p-JNK (4671s, Cell Signaling Technology), P38 (9212s, Cell Signaling Technology), p-P38 (4511s, Cell Signaling Technology), AKT (4691,Cell Signaling Technology), p-AKT (4060, Cell Signaling Technology), STAT3 (9132, Cell Signaling Technology), and p-STAT3 (9131, Cell Signaling Technology). Corresponding secondary antibodies conjugated to horseradish peroxidase were used for detection. Staining was detected using chemiluminescence and quantified by Image Lab software (Bio-Rad). All expression data was provided relative to GAPDH (diluted 1:500, Aspen) staining for the same samples on the same gels.
10
Western Blotting Analysis of Cellular Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed in RIPA buffer (Cell Signalling Technology, Danvers, MA), and the resultant proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PolyScreen membranes (NEN, Boston, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and probed with antibodies targeting the following proteins: β-catenin (Cell Signalling Technology), cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AXIN2, c-Myc, STAT3, cyclin B1, Gab1, c-caspase-3, p-ERK (Cell Signalling Technology), HA, β-actin, and γ-tubulin (Santa Cruz Biotechnology). Primary antibodies were detected with HRP-conjugated anti-mouse or anti-rabbit antibodies, as appropriate, that were subjected to enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
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!