The cell lines used were MiaPaca2. Neo, MiaPaca2. MUC1, CFPAC, HPAFII. control siRNA, and HPAFII. MUC1siRNA. Cells were treated with either 2 μM Napabucasin (Selleckchem, USA) or the vehicle (phosphate buffer saline) for 48 h. Cell lysates were prepared and western blotting performed as previously described34 . Membranes were blocked with commercial blocking buffer (Thermo Fisher) for 30 min at room temperature and incubated with primary antibodies overnight at 4 °C. The antibodies used were: Armenian hamster monoclonal anti-human MUC1 cytoplasmic tail (CT2) antibody (1:500). MUC1 CT antibody CT2 was originally generated at Mayo Clinic and purchased from Neomarkers, Inc. (Portsmouth, NH)38 (link). CT2 antibody recognizes the last 17 amino acids (SSLSYNTPAVAATSANL) of the cytoplasmic tail (CT) of human MUC1. Membranes were also probed with the following antibodies from Cell Signaling Technology (1:1,000), p-STAT3 (Y705), total STAT3, β-actin, and from ABclonal (1:1000) p-STAT3 (S727) and β-actin. Densitometric analysis was conducted using the ImageJ software. First, each density unit for the particular protein was normalized to their respective β-actin density and then represented as phospho/total.
P stat3
Manufactured by ABclonal
Sourced in United States
P-STAT3 is a lab equipment product from ABclonal, designed for the detection and quantification of phosphorylated STAT3 (Tyr705) in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by ABclonal (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fibronectin, by ABclonal (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Proliferating cell nuclear antigen (pcna), by ABclonal (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Claudin 1, by ABclonal (1 mentions)
Notopterol, by Chengdu Biopurify Phytochemicals (1 mentions)
Hrp labeled goat anti rabbit secondary antibody, by Proteintech (1 mentions)
Stat3, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Bca protein concentration determination kit, by Dingguo (1 mentions)
9 protocols using p stat3
1
Napabucasin Modulates MUC1 and STAT3 Signaling in Pancreatic Cancer
2
Notopterol-mediated Regulation of Cellular Signaling
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Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was supplied by Hyclone (Logan, UT, USA). An FITC Annexin V kit was obtained from Elabscience Biotechnology (Houston, TX, USA). Antibodies specific to ERK1/2, p-c-Jun, p-p65 (p-NF-κB), p-Akt, Cyclin E, CDK4, N-cadherin, Vimentin, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), whereas antibodies specific to p-ERK, p38, p-p38, STAT3, p-STAT3, Akt, c-Jun, MCM2, PCNA, Cylin D1, E2F, Fibronectin, ZEB-1, MT1-MMP, uPA, and Claudin-1 were purchased from Abclonal (Woburn, MA, USA). Antibodies for the detection of Ki67, Snail, and p65(NF-κB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellulose membrane and ECL reagent were supplied by GE Healthcare (Little Chalfont, UK). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA). Notopterol with purity of 98% was ordered from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).
3
Western Blot Analysis of Stem Cell Markers
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Western blotting was used to detect the target protein in the sample. The total protein content in the sample was detected in a 96-well plate using a BCA protein concentration determination kit (Dingguochangsheng). Protein tracer sample buffer (reduction, 5×; CWBIO) was mixed with protein samples in a ratio of 1:4. The mixtures were then placed in a boiling water bath for 3 minutes. The samples were cooled to room temperature and centrifuged at 13 000×g at 4 °C for 30 seconds. Denatured proteins were directly loaded to a sodium dodecyl sulfate-page gel, and conventional electrophoresis (concentration gel voltage 60 V, separation gel voltage 120 V) and membrane transfer (current 200 mA) were performed using a Bio-Rad protein imprinting device. After completion, the transferred films were incubated at 4 °C overnight. The rabbit primary antibodies used were STAT3 (Proteintech), pSTAT3 (Tyr705; ABclonal), KLF4 (Proteintech), OCT4 (Proteintech), and β-actin (Proteintech). Samples were incubated with HRP-labeled goat anti-rabbit secondary antibody (Proteintech) at room temperature for 1 hour, washed with 1× TBST 3 times for 10 minutes each, and finally ECL development was performed.
4
Western Blot Analysis of Inflammatory Mediators
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer on ice with protease and phosphatase inhibitors (Calbiochem). The total protein concentration was determined with a BCA Protein Assay Kit (CWBIO). Equal amounts of protein were separated by 12% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). The membranes were blocked with TBST containing 5% non‐fat milk and incubated with primary antibodies against C5a (Abcam), C5aR (Abcam), IL‐1β (Abclonal), MCP‐1 (Abcam), STAT3 (Abclonal), p‐STAT3 (Abclonal) and β‐actin (Santa Cruz) overnight at 4°C. After washes with TBST, the PVDF membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz) at 37°C for 1 hour. The protein bands on the PVDF membranes were visualized with an enhanced chemiluminescence kit (Millipore) and quantified by NIH ImageJ software, and the protein levels were normalized to those of β‐actin.
5
Immunomodulatory Mechanisms of CFA-Induced Inflammation
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Complete Freund’s adjuvant (CFA), methotrexate, and TRAP staining kit were both procured from Sigma-Aldrich (St. Louis. MO). ELISA analysis kits for genes TNF-α, IL-1β, IL-6, and IL-17 were bought from PeproTech (Rocky Hill, USA). Dulbecco’s modified Eagle’s medium (DMEM) along with fetal bovine serum (FBS) and antibiotic solution were all obtained from Gibco BRL (Grand Island, NY, USA). FITC-conjugated monoclonal CD90.2 antibody was purchased from BioLegend (San Diego, CA, USA). JAK-1, p-JAK-1, JAK-3, p-JAK-3, STAT-3, p-STAT-3, RANKL, OPG primary antibodies were purchased from ABclonal (Woburn, MA, USA), and secondary antibodies conjugated with HRP and FITC were obtained from Cell Signaling Technology (Beverly, MA, USA). 4′, 6-diamidino-2-phenylindole (DAPI) was obtained from Thermo Fischer Scientific (Waltham, Massachusetts, USA). All other reagents and solvents for experimental use were of analytical standard procured from local commercial sources.
6
STAT3 Protein Expression Analysis in U937 Cells
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Total protein was extracted from U937 cells by RIPA kit, and the protein concentration was determined by BCA kit (cat no. P0012, Beyotime Biology). Polyacrylamide gel electrophoresis was performed at 40 μg/lane. After protein isolation, the protein was transferred to the PVFD membrane by wet transfer method and sealed with 5% skim milk at room temperature for 2 h. Primary antibody STAT3(cat no. A19566, ABclonal) and p‐STAT3 (cat no. AP0715, ABclonal) diluted at 1:1000 were added, respectively, and incubated at 4°C overnight. The PVFD membrane was 3 times washed by TBST, and HRP labeled sheep anti‐rabbit IgG antibody was added to incubate at room temperature for 1 h. After rinsing with TBST, added ECL reaction solution and photographs were taken using a UVP gel imaging system. The relative protein expression level was calculated as the optical density value ratio of the target band to the inner reference band (β‐actin). The experiment was repeated three times for each group.
7
Western Blot Analysis of Signaling Proteins
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The total proteins extracted from the tissue were obtained using RIPA buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China), which contained PMSF (Beyotime Biotechnology Co., Ltd.) to inhibit protease degradation. Next, the samples were separated via electrophoresis on an SDS-PAGE gel (Solarbio Biotechnology) and then transferred onto polyvinylidene fluoride membranes (Millipore, Boston, Massachusetts, USA). Afterward, the membranes were incubated with one of the primary antibodies (IL-11Rα, 1:500 dilution, HuaBio Biotechnology Co., Ltd.; GP130, 1:500 dilution, ABclonal, Wuhan, China; NSUN5, 1:500 dilution, Thermo Fisher, Waltham, Massachusetts, USA; JAK2, 1:1000 dilution, ABclonal; P-JAK2, 1:500 dilution, ABclonal; STAT3, 1:1000 dilution, ABclonal; P-STAT3, 1:500 dilution, ABclonal; CD3, 1:1000 dilution, HuaBio Biotechnology Co., Ltd.; and β-actin, 1:5000 dilution, HuaBio Biotechnology Co., Ltd.) overnight at 4°C. Next, a horseradish peroxidase-labeled secondary antibody (Thermo Fisher) was added and incubated for 1 h at room temperature. Lastly, the protein bands were developed using a chemiluminescence detection kit (Millipore, Bedford, MA, USA), and the relative intensities of the bands were determined using Quantiscan software (Biosoft, Cambridge, United Kingdom).
8
Protein Expression Analysis in Mouse Lung and HPASMC
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Total proteins were extracted from homogenized mouse lung tissues or HPASMCs and the protein concentration was measured using a BCA protein concentration assay kit (Beyotime Institute of Biotechnology, China). The protein samples were separated by SDS‐PAGE and transferred to PVDF membranes, followed by blocked with 5% skim milk powder solution (Inner Mongolia Yili Industrial Group Co., Ltd., China)/3% BSA (Labgic Technology Co., Ltd., China) for 1 h. The molecular weights of protein blots can be distinguished by the loading protein marker. The PVDF membranes were cut according to the protein marker prior to hybridization with primary antibodies. Then membranes were incubated overnight at 4 °C with primary antibodies against SOCS5 (A7952, 1:1,000), JAK2 (A11497, 1:1,000), p-JAK2 (AP0531, 1:1,000), STAT3 (A1192, 1:1,000), p-STAT3 (AP0705, 1:1,000, all from ABclonal Technology Co., Ltd., China), β-actin (sc-47778, 1:1,000, santa cruz biotechnology, inc., USA) and incubated with HRP (1:5,000, Beyotime Institute of Biotechnology, China) bound goat anti-rabbit IgG (A0208)/goat anti-mouse IgG (A0216) at 37 °C for 45 min. All antibodies were diluted with 5% skim milk powder solution. Finally, the protein bands were detected by the ECL system (Beyotime Institute of Biotechnology, China).
9
Western Blot Analysis of Protein Signaling Pathways
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Total protein was extracted using RIPA lysis buffer (20-188, Millipore) supplemented with protease inhibitor (ab201120, Abcam) and quantified using the bicinchoninic acid kit (23225, ThermoFisher). Equivalent amount of proteins was separated using 10% SDS–PAGE and transferred onto PVDF membranes (IPVH00010, Millipore). After blocking with 5% BSA (A3059, Sigma), the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were PCNA (A0264, Abclonal, Massachusetts, USA), FGF21 (A3908, Abclonal), FGFR1 (A21219, Abclonal), FGFR2 (A19051, Abclonal), FGFR3 (A19052, Abclonal), FGFR4 (A9197, Abclonal), KLB (A15629, Abclonal), p-FGFR (3471S, Cell Signaling Technology), STAT3 (4904, Cell Signaling Technology), p-STAT3 (AP0705, Abclonal), FoXO1 (A2934, Abclonal), p-FoXO1 (AP0172, Abclonal), Akt (ab179463, Abcam), p-Akt (ab192623, Abcam), Bax (A0207, Abclonal), Bcl-2 (A19693, Abclonal), Bcl-xl (A0209, Abclonal), α-Tubulin (AC007, Abclonal), β-Actin (ab8226, Abcam) and GAPDH (AC002, Abclonal). The membranes were then incubated with secondary antibodies (7074, 7076, Cell Signaling Technology, Massachusetts, USA) for 1 h at room temperature. Protein bands were visualized using an HRP substrate (WBLUF0500, Millipore) on a gel imaging system (ChemiDoc XRS+, Bio-Rad, California, USA). The images were analyzed using Image Lab software (version 6.1, Bio-Rad).
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