Differentiated cells were harvested and lysed in RIPA buffer supplemented with proteinase inhibitor cocktail (MedChemExpress). Proteins were separated on 10% SDS-PAGE gel and electro-transferred to PVDF membranes (Millipore). The membranes were blocked in blocking solution (5% skim milk in PBS) for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against JAK2 (1:1000, Cell Signaling Technology), p-JAK2 (Tyr1007/1008, 1:1000, Abcam), STAT3 (1:2000, Cell Signaling Technology), p-STAT3 (Tyr705, 1:2000, Abcam), HIF-1a (1:500, Santa Cruz Biotechnology), cyclin D1 (1:5000, proteintech), c-Myc (1:1000, Abmart), GAPDH (1:2000, Affinity) and β-actin (1:3000, Affinity). Thereafter, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-linked secondary antibodies (1:2000, Wanleibio). The protein bands were detected by using an enhanced chemiluminescence kit (Wanleibio).
P stat3
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany
P-STAT3 is a lab equipment product that is used to detect and quantify the phosphorylated form of the STAT3 protein. STAT3 is a transcription factor that plays a key role in various cellular processes, and its phosphorylation is an important indicator of its activation. The P-STAT3 product can be used in a variety of applications, including cell signaling studies, drug discovery research, and protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Abcam (68 mentions)
P jak2, by Abcam (36 mentions)
Gapdh, by Abcam (25 mentions)
β actin, by Abcam (25 mentions)
Pvdf membrane, by Merck Group (16 mentions)
Gapdh, by Cell Signaling Technology (15 mentions)
P akt, by Abcam (11 mentions)
Bcl 2, by Abcam (10 mentions)
E cadherin, by Abcam (10 mentions)
P akt, by Cell Signaling Technology (10 mentions)
Bcl2 associated x (bax), by Abcam (9 mentions)
Interleukin 6 (il 6), by Abcam (8 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Vimentin, by Abcam (7 mentions)
Gapdh, by Proteintech (7 mentions)
Stat3, by Cell Signaling Technology (6 mentions)
N cadherin, by Abcam (6 mentions)
Cyclin d1, by Abcam (6 mentions)
Stat3, by Proteintech (6 mentions)
184 protocols using p stat3
1
Western Blot Analysis of JAK-STAT Pathway
2
Protein Extraction and Analysis from Ventricular Muscle
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Put ventricular muscle tissue into EP tube and put it on ice, then add 300 μ L RIPA protein lysate and protease inhibitor into each tube, then sterilize and wipe the tissue with scissors, and cut the tissue as much as possible with scissors, and keep it on ice during the cutting process. For the extraction of protein from adherent parietal cells, add an appropriate amount of protein lysate into the cell culture dish and transfer the protein lysate to 1.5 ml EP tube and place it on ice. After testing the protein concentration using BSA standards, add 5 X Loading Buffer protein loading buffer, mix well, and then boil in a 99℃ metal bath for 10 minutes. Cool on ice and use for Western blot experiments. Protein was separated by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane. After 2h BSA blocking, the membranes were cultured with primary antibodies of p-JAK2, JAK2, p-STAT3, STAT3, Bax, Bcl-2, caspase-3 and β-actin (1:1000, Abcam, MA, USA) overnight at 4 °C. After incubating the second antibody for 2 hours, prepare the developer according to the ECL developer instructions and perform exposure development. Use a chemiluminescence imager (ProteinSimple, San Jose, CA, USA) to expose the bands and collect statistical image results.
3
Protein Extraction and Western Blot Analysis
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The total protein of EOC tissues and cell lines was isolated via a cell protein extraction kit (KeyGen Biotechnology, Nanjing, PR China) according to the manufacturer's instructions. The BCA kit (Beyotime Biotechnology, Beijing, PR China) was used to determine the concentration. An equivalent amount of protein from each sample was separated by 4 to 20% SDS-PAGE (Genscript, Nanjing, China), transferred to a nitrocellulose membrane and blocked with 2% bovine serum albumin (KeyGen Biotechnology). The membrane was then incubated withprimary antibodies against NPTX2, IL6, p-JAK2, JAK2, p-STAT3, STAT3 and β-actin (Abcam Technology, Cambridge, UK) at 4 °C overnight, followed with TBST washing and incubated with secondary antibody. The ECL kit (Beyotime) was used to detect the bands on each membrane, and IMAGE J software (National Institutes of Health, Bethesda, MD, USA) was used for quanti cation.
4
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical staining was performed according to a previous description (5 (link)). Paraformaldehyde-fixed tumor sections (4-µm in thickness) were incubated with rabbit-anti-human monoclonal antibodies p-STAT3 (1:100 dilution; product code ab76315; Abcam), E-cadherin (1:500 dilution; product code ab227639, Abcam), N-cadherin (1:150 dilution; cat. no. 13116; Cell Signaling Technology, Inc.) and vimentin (1:200 dilution; product code ab92547; Abcam), followed by incubation with horseradish peroxidase-conjugated goat-anti-rabbit secondary antibody (1:500 dilution; cat. no. WLA023; Wanleibio Co., Ltd.). The sections were then incubated with developing solution and counterstained with hematoxylin. The color was fixed with acid alcohol and dehydration steps. Finally, the sections were observed with a light microscope (magnification ×100), and the representative images were photographed. The experiment was performed in triplicate.
5
Western Blot Analysis of Protein Expression
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Cell and tissue samples were lysed with radio‐immunoprecipitation assay buffer (RIPA) with protease and phosphatase inhibitor cocktail (Promega, Fitchburg, WI, USA). Proteins were separated by SDS/PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes by the Trans‐Blot System (Bio‐Rad, Hercules, CA, USA). The membranes were blocked by milk and then incubated with specific primary antibodies against Cyr61 (Abcam, Cambridge, MA, USA, 1 : 1000), integrin β5 (Cell Signaling Technology, Danvers, MA, USA, CST, 1 : 1000), FAK (CST, 1 : 1000), p‐FAK (CST, 1 : 1000), P65 (CST, 1 : 1000), GAPDH (Abcam, 1 : 1000), MEK (CST, 1 : 1000), p‐MEK (CST, 1 : 1000), ERK (CST, 1 : 1000), p‐ERK (CST, 1 : 1000), STAT3 (CST, 1 : 1000), p‐STAT3 (CST, 1 : 1000), MMP2 (Abcam, 1 : 2000), and HIF‐1α (Abcam, 1 : 500). Finally, membranes were incubated with a specific secondary antibody and visualized by ECL Blotting Detection Reagents. GAPDH served as a control for western blot analysis.
6
Immunohistochemical Analysis of ANXA2 and p-STAT3
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Fresh intracranial tumor tissues from nude mice were fixed with 4% paraformaldehyde and then embedded in paraffin. Sections were incubated at 4 °C overnight with primary antibodies against ANXA2 (1:1000; Abcam) and p-STAT3 (1:500; Abcam). Sections were then incubated with secondary antibody (1:1000; Santa Cruz) for 2 h at room temperature and stained with diaminobenzidine until brown granules appeared.
7
Protein Extraction and Western Blot Analysis
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Before harvest, BV-2 cells were washed with cold PBS and then lysed with lysis buffer containing protease inhibitors for 30 min on ice. The samples were centrifuged at 12000 rpm, 4°C for 15 min. Then, the protein concentrations were determined by using a BCA protein assay kit (Beyotime Insititute of Biotechnology, Haimen, China). Proteins were electrophoresed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE Bio-Rad, CA, USA) and transferred electrophoretically to PVDF membranes. Then, the membranes were blocked with 5% skim milk at room temperature (RT) for 1 h and were incubated with primary antibodies overnight at 4°C. Subsequently, membranes were washed and incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, membranes were washed and detected with enhanced chemiluminescence. Primary antibodies were as follows: anti-β-tubulin (1 : 2000; Sangon Biotech, China), anti-REST (1 : 1000; Abcam, USA) [8 (link)], anti-lambin 1 (1 : 2000, Proteintech, China), Jak2 (1 : 5000, Abcam, USA), p-JAK2 (1 : 1000, abcam, USA), STAT3 (1 : 1000, Abcam, USA), p-STAT3 (1 : 1000, Abcam, USA), PGRN (1 : 1000; R&D systems, USA), and lamin B1 (1 : 1000, Abcam, USA).
8
Protein Expression Analysis by Western Blotting
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Protein lysates were harvested using RIPA lysis buffer with freshly added protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Millipore, Bedford, USA), which were then blocked with 5% skim milk and incubated with RNF7 (ab181986; 1:5000), SOCS1 (ab62584; 1:600), HK2 (ab227198; 1:8000), STAT3 (ab119352; 1:2000), p-STAT3 (ab76315; 1:5000; all from Abcam), GLUT1 (orb157188; 1:2000; Biorbyt, St Louis, MO, USA), cleaved PARP1 (#5625; 1:1000), cleaved caspase-3 (#9661; 1:1000), and GAPDH (#5174; 1:2000; all from Cell Signaling Technology, Danvers, MA, USA) primary antibodies and HRP-conjugated secondary antibodies (A0208, A0181; 1:1000; Beyotime). Protein bands were visualized using an enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). Protein levels were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
9
Immunofluorescence Staining of p-STAT3 and CD31
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Cells were firstly fixed and incubated with the p-STAT3 (1:100; Abcam) or CD31 (1:100; Abcam) antibody (Cell Signaling Technology, 1:500) overnight at 4°C, as previously described. After being washed three times, these cells were sequentially incubated with Fluorophore-conjugated secondary antibody (Alexa Fluor 488 or 546; Invitrogen, 1:200) at room temperature for 60 min. Cell nuclear was stained with DAPI nuclear antibody (Invitrogen, 1 mM) at room temperature for 15 min. The fluorescence was captured using a confocal microscope (Olympus) [25 (link)].
10
Histopathological Analysis of Colon Tissue
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Colon tissue samples were flushed with PBS and fixed in 4% formalin at 4 °C overnight. After fixation, tissue samples were dehydrated and immersed in xylene and embedded in paraffin. 1–2 μm paraffin sections were cut, dewaxed and stained histochemically with hematoxylin and eosin or immunohistochemically with the respective antibodies (pSTAT3 (Clone: EP2147Y; Abcam; Cat #: ab76315), CD3 (DAKO; Cat #: N1580), KI67 (clone: TEC3; DAKO; Cat #: M7249), Casp3 (Clone: Asp175; Cell Signaling; Cat #: 9664 S) and F4/80 (Clone: BM8; Thermo Fischer Scientific; Cat #: 14-4801), followed by Streptavidin-AP kit (DAKO; Cat #: K5005) and Envision PO kit (DAKO; Cat #: K5007). The sections were processed, stained and analyzed in a blinded fashion. Representative pictures were taken by a pathologist and were provided further to the scientist for decoding and illustrations of disease characteristics. The degree of colonic inflammation was scored using a previously described scoring system by Erben and colleagues56 (link).
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