The design and synthesis of HJC0152 were described in our previous publication.19 Antibodies against STAT3 (Cat# 4904), STAT1 (Cat# 9172), STAT5 (Cat# 9363), γ‐H2AX (Cat# 9718) and Alexa Fluor 555 goat anti‐rabbit immunoglobulin G (H + L) (Cat# 4413) were procured from Cell Signaling Technology. Bcl2 (Cat# 12789‐1‐AP) and GAPDH (Cat# HRP‐60004) antibodies were obtained from Proteintech. An antibody against STAT3 phosphorylated at tyrosine residue 705 (p‐STAT3(Tyr705)) (Cat# ab76315) was purchased from Abcam. ProLong Gold antifade reagent with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Cat# P36941) was obtained from Thermo Fisher Scientific. All other reagents used were purchased from commercial sources unless otherwise indicated. All reagents were dissolved and used as recommended by their suppliers.
P stat3 tyr705
Manufactured by Abcam
Sourced in United States
P-STAT3 (Tyr705) is a recombinant protein that corresponds to the phosphorylated form of STAT3 (Signal Transducer and Activator of Transcription 3) at tyrosine 705. This protein is commonly used as a reference standard or positive control in various applications, such as Western blotting and ELISA, to detect and quantify the phosphorylation of STAT3 at this specific site.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Abcam (4 mentions)
Gapdh, by Abcam (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
P stat3 ser727, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Image acquisition and analysis software, by Analytik Jena (1 mentions)
Socs3, by Abcam (1 mentions)
Sm22α, by Abcam (1 mentions)
P smad3, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Smad3, by Abcam (1 mentions)
Dab chromogen kit, by Vector Laboratories (1 mentions)
Alexa fluor 488, by Cell Signaling Technology (1 mentions)
11 protocols using p stat3 tyr705
1
Antibody-based Protein Expression Analysis
2
Protein Expression Analysis in Rheumatoid Arthritis Synovial Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RASFs (n = 5) were collected and dissolved using 200 μL RIPA buffer (Beibo, China) with 2 mM protease inhibitors sodium orthovanadate (Beibo, China) and 2 μL phosphatase inhibitors (Beibo, China) for 30 min on ice and then centrifuged at 10,000 rpm for 30 minutes at 4 °C. After, the total protein concentrations were measured using the BCA method (Biyuntian, China). Equal amounts (30 μg) of protein were separated by 10% sodium dodecyl sulfate denatured polyacrylamide gel and transferred onto poly (vinylidene fluoride) membranes in bovine serum albumin in Tris-based saline with Tween 20 for 1 hour at room temperature. Membranes were incubated with rabbit antihuman antibodies at the recommended dilution [STAT3 at a dilution of 1:100 (Abcam, USA), p-STAT3 (Tyr705) at 1:10,000 (Abcam, USA), SOCS3 at 1:10,000 (Abcam, USA), GAPDH at 1:5000 (Abcam, USA)] overnight at 4 °C. After washing in Tris-based saline with Tween 20, the membranes were further incubated with a secondary anti-rabbit antibody (1:10,000) for 2 hours. Enhanced chemiluminescence solution was added onto the membranes, and protein expression was quantified using the Laboratory Work Image Acquisition and Analysis Software (UVP, Upland, CA). GAPDH was used as a loading control.
3
Comprehensive Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We purchased the following primary antibody from Cell Signaling Technology: PKM2 (1:1,000), snail (1:500), E‐Cadherin (1:500), HK1 (1:1,000), p‐STAT3 (Tyr705) (1:1,000), STAT3 (1:1,000), HK2 (1:1,000); vimentin (1:2,000), α‐SMA (1:1,000), SM22α (1:800), SMAD3 (1:500), p‐SMAD3 (1:500) were from Abcam, HIF‐1α (1:200) was purchased from Novus Biologicals.
4
Immunohistochemical Evaluation of SHP-1 and p-STAT3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For tissue microarray and immunohistochemistry, the procedures followed our previous methods and the manufacturer’s instructions [35] (link). SHP-1 and p-STAT3Tyr705 antibodies were purchased from Abcam (Cambridge, MA). Omission of the primary antibody served as a negative control. Immunopositive results were evaluated by 2 pathologists. The intensity of stained cells was scored as 0, 1, 2, or 3. Percentages of stained cells were counted. A final immunohistochemical score (H-score) was calculated by summing the products of the staining intensities (0-3) and distributions (0%-100%). H-scores ranged from 0 to 300. An H-score equal to or greater than 150 was defined as strongly positive for SHP-1 staining; all others were scored as weakly positive. An H-score greater than 10 was defined as strongly positive for p-STAT3Tyr705 staining (nuclear staining); all others were scored as weakly positive.
5
Immunohistochemistry and Immunofluorescence Staining of HNSCC Samples
6
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed in ice-cold RIPA lysis buffer with added protease and phosphatase inhibitor (Cell Signaling Technology, Danvers, MA USA). Equal amounts of protein were resolved and separated on an SDS/PAGE gel (BioRad TrisGlycine 4–20% gel), transferred onto PVDF membranes, and subjected to immunoblot analyses. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, and were incubated with primary and secondary antibodies according to the manufacturer’s instructions. Blotting was performed using antibodies targeting PLK1 (Abcam, Cambridge, UK; Cat. #: 17056), DNA methyltransferase 1 (DNMT1) (BD Biosciences, San Jose, CA USA; Cat. #: 612618), Bcl-xL (Cell Signaling; Cat. #: 2764), Cleaved PARP (c-PARP) (Cell Signaling; Cat. #: 9541), p53 (DO-1) (Santa Cruz, Cat. #126), p-p53 (Ser15) (Cell Signaling; Cat. #: 9284), STAT3 (BD Biosci, Cat. #: 610189), p-STAT3 (Tyr705) (abcam; Cat. #: 76315), Cleaved Caspase-7 (c-Caspase-7) (Cell Signaling; Cat. #: 9491), Cleaved Caspase-3 (c-Caspase-3) (Cell Signaling; Cat. #: 9541), and Vinculin (Cell Signaling; Cat. # 4650). Dilutions used for Cleaved Caspase-3 and Cleaved Caspase-7 were 1:250, and for other antibodies were according to the company’s recommendations.
7
Molecular Pathway Analysis in Joint Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Joint tissues were incubated overnight at 4 °C with primary antibodies against SMILE (Abcam, Cambridge, UK), B-cell activating factor receptor (BAFF-R) (Abcam), pAMPK (Abcam), mTOR (Cell Signaling, MA, USA), pSTAT3 ser727 (Abcam), Pstat3 Tyr705 (Abcam), IL-1β (Abcam), IL-6 (Abcam), IL-17(Abcam). Subsequently, samples were incubated with a biotinylated streptavidin–peroxidase complex for 1 h, and the signals were developed using chromogen 3,3′- diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined under a photomicroscope (Olympus, Tokyo, Japan). The number of positive cells in high-power digital images (magnification, ×400) was counted using Adobe Photoshop software (Adobe, San Jose, CA, USA). Stained cells were counted independently by three observers, and the mean values were evaluated.
8
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were made in RIPA buffer and subjected to SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) and incubated with primary antibody, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Specific proteins were detected using enhanced chemiluminescence reagent. The primary antibodies including p-STAT3Tyr705, STAT3, SHP-1, Vimentin, Fibronectin, E-cadherin and GAPDH were purchased from Abcam (Cambridge, MA). For quantification of protein levels on chemiluminescent western blots, densitometry Image J software was employed. Statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).
9
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Groups of cells were harvested and were lysed with RIPA (Beyotime, Ningbo, China) for 30 min. After centrifugation, the supernatant was collected. After quantification, the protein was denatured, separated with 10% SDS-PAGE by electrophoresis, and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After closure with 5% skim milk powder for 2 h, the membranes were exposed to diluted primary antibodies at 4°C overnight and diluted secondary antibodies (Abcam, Cambridge, MA, USA) for 1.5 h. The protein bands on the membranes were tested by ECL kit, and the protein level was analyzed by graphing. The primary antibodies (p-Stat3 (Tyr 705) and Stat3) were from Abcam (Cambridge, MA, USA); JAK2, p-JAK2 (Tyr 1007), CDK4, Bcl-2, and GAPDH were from Cell Signaling Technology (Beverly, MA, USA); and MMP-9 was from Affinity (JiangSu, China).
10
Immunoblotting Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against STAT3, P-STAT3 (Tyr705), AKT, P-AKT, C-MYC, Cyclin D1, Cleaved-Caspase-3, HGF, MEKK2, Survivin, and Bcl-2 were purchased from Abcam (Cambridge, MA, USA). CPT was purchased from Selleckchem (Houston, TX, USA), and dissolved in dimethyl sulfoxide (DMSO; 10 mM) for subsequent experiments.
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!