3-(4,5-DimethylthiazoL-2-yl)-2,5-diphenyltetrazolium bromide(MTT) was purchased from Sigma Chemical Co. (St. Louis, USA). Annexin V-FITC/PI Apoptosis Kit, TUNEL Apoptosis Kit, and Ki-67 Immunohistochemical Monitoring Kit were purchased from Proteintech Group, Inc. (Chicago, USA). Antibodies against β-actin, STAT3, p-STAT3, and VEGF were also purchased from Proteintech Group, Inc. (Chicago, USA). Cisplatin (DDP) was purchased from Qilu Pharmaceutical Company (Ji'nan, China).
P stat3
Manufactured by Proteintech
Sourced in United States, China
P-STAT3 is a primary antibody that detects phosphorylated STAT3 protein. It is suitable for use in Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Proteintech (6 mentions)
Erk1 2, by Proteintech (3 mentions)
P akt, by Cell Signaling Technology (2 mentions)
β actin, by Proteintech (2 mentions)
P jak1, by Proteintech (2 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (2 mentions)
α sma, by Proteintech (2 mentions)
Cisplatin ddp, by Qilu Pharmaceutical (1 mentions)
Fluorescein isothiocyanate conjugated anti human cd4, by BD (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Anti human cd28, by R&D Systems (1 mentions)
P mapk, by Cell Signaling Technology (1 mentions)
Mouse igg1, by BD (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Allophycocyanin apc conjugated anti human cd25, by BD (1 mentions)
Gapdh, by Proteintech (1 mentions)
Mouse igg2a, by BD (1 mentions)
P jak2, by Proteintech (1 mentions)
Hu6 mcs cmv puromycin, by Genechem (1 mentions)
Wp1066, by Selleck Chemicals (1 mentions)
11 protocols using p stat3
1
MTT Assay for Cell Viability
2
Multiparameter Immune Cell Analysis
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The following antibodies (to human) were from BD Biosciences (Fremont, CA, USA): fluorescein isothiocyanate conjugated anti-human CD4, phycoerythrin (PE)-conjugated anti-human CD4 and IL-17A, allophycocyanin (APC)-conjugated anti-human CD25, phycoerythrin-Cy7-conjugated anti-human IFN-γ, IL-4, and their isotype-matched control antibodies (mouse IgG1, mouse IgG2a). The following antibodies were from eBioscience (San Diego, CA, USA): PE-Cy7-conjugated Foxp3. Purified anti-human CD3, anti-humanCD28, anti-human IL-6 antibodies and recombinant IL-6 antibodies were all from R&D Systems (Minneapolis, MN, USA). AKT inhibitor (GSK690693), p38/MAPK inhibitor (SB203580) and STAT3 inhibitor (NSC74859) were from Sigma-Aldrich (St. Louis, MO, USA). Human IL-6, TGF-β ELISA kits were from BioLegend (San Diego, CA, USA). The following antibodies (to human) were from Cell Signaling technology (Inc., USA): STAT3, AKT, MAPK, p38, p-ERK1/2, p-MAPK, p-AKT, p-p38. The following antibodies (to human) were from Proteintech (Inc., China): ERK1/2, ERK1/2, p-STAT3, and GAPDH.
4
Targeting JAK2/STAT3 Pathway in Cells
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The JAK2/STAT3 inhibitor WP1066 was purchased from Selleck Chemicals (Boston, MA, USA). Antibodies for p-JAK, JAK, p-STAT3, STAT3, ATP7A were purchased from Proteintech Group (Wuhan, China). SLC30A7 overexpression plasmids and short hairpin RNA (shRNA) were produced by GV112 vector (hU6-MCS-CMV-Puromycin; GeneChem, China). On the basis of the manufacturer’s recommendation, lentiviral vectors expressing shRNA or scrambled transfected into cells. Steady cell clones transfected with shRNA expressing constructs were chosen with puromycin intervention after infection.
5
Investigating mTOR Pathway Regulation in AML
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AML cells were cultured in complete medium containing 10% serum, stimulated with 25 ng/ml IL6 for 30 min. Cells were harvested, washed with 1 × PBS and lysated with 1 × RIPA lysis buffer containing protease/phosphatase inhibitors (Thermo Fisher #78,441). The lysates were sonicated and centrifuged at 14,000 g for 15min. Western blot analysis was performed with the following primary antibodies, including anti-4EBP1 (CST#9644S), p-4EBP1 (CST#2855P), p-mTOR (CST#5536S), mTOR (CST#2983S), p-STAT3 (CST#9145S), STAT3 (CST#4904S), p-P70S6K (CST#9205S), P70S6K (CST#35,708), CDK6 (Proteintech#14,052), CCND1 (Abcam#ab54503), GAPDH (CST#5174), and HRP conjugated secondary antibodies (Abcam#ab205718, Abcam#ab205719). Detection was conducted using a chemiluminescence substrate (Omics Bio), and images were acquired using ImageQuantTM LAS 4,000 camera and quantified using lmageJ software version1.53 (NIH, Bethesda, MD, USA). To examine whether the phosphorylated protein levels of mTOR and its downstream effectors (P70S6K and 4EBP1) were significantly inhibited, we normalized the levels of phosphorylated protein to the corresponding total protein. Uncropped images for the blots were provided in Additional file 12 .
6
Comprehensive Protein Analysis via Western Blot
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Lysate (Beyotime P0013J) containing protease inhibitor and EDTA was utilized to lyse tissues and cells. Protein was then separated by 10% PAGE gels (Epizyme PG112) and transferred onto PVDF membranes (Millipore IPVH00010). The PVDF membranes were blocked with 10% bovine serum albumin (BSA) and incubated with primary antibodies β-actin (CST 3700S), A1AT (Proteintech 16382-1-AP), c-myc (SANTA sc-40), lamin B1 (CST13435S), p-JAK1 (CST 3331S), JAK1 (CST 3332S), p-STAT3 (CST 9145S), STAT3 (CST 4904S) and CEBPB (Proteintech 23431-1-AP) at 4 °C overnight. Then, the membranes were incubated with secondary antibody (Zsbio zb-2301 and Zsbio zb-2305) at room temperature for 1 h.
7
Western Blot Analysis of Cellular Signaling
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The cells were lysed in radioimmunoprecipitation assay buffer (Beyotime) in the presence of protease inhibitors (Roche). Whole-cell lysates were prepared and subjected to 10% SDS-PAGE (GeneStar) and transferred to polyvinylidene fluoride membrane (Bio-Rad). The membranes were probed overnight at 4°C with the antibodies specific for TIPE2 (Proteintech), ARG1, iNOS, p-STAT3 (Tyr705), STAT3, p-ERK (p-Erk1/2, Thr202/Tyr204), ERK (Erk1/2), p-AKT (Ser473), AKT, p-GSK-3β (Ser9), GSK-3β, p-C/EBPβ (Thr235, Thr188, and Thr47), p-threonine (Cell Signaling Technology), p47phox, C/EBPβ, and β-actin (Santa Cruz), at a 1:1,000 dilution (all antibodies shown in Table S2 ). Membranes were washed and incubated for 1 h at room temperature with secondary antibodies conjugated with peroxidase. Membrane-bound immune complexes were detected using Super ECL detection reagent (Applygen) on an Amersham Imager 600 (GE Healthcare).
8
Proteomic Analysis of Signaling Pathways
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Cells were lysed on ice using lysis buffer as instructed before27 ; then the lysate proteins were collected and further analyzed. Protein A/G agarose or Ni‐NTA beads were used for immunoprecipitation or pulldown assays, respectively. Immunoblotting was performed according to standard method with primary antibodies against pY antibody (Abcam EPR16871 ), pEGFR (CST#3777S), EGFR (CST#4267), pSTAT3 (CST#4113), STAT3 (CST#9139), CK8 (17514‐1‐AP, Proteintech), CD71 (ABclonal#A5865), Vimentin (10366‐1‐AP, Proteintech), GRB2 (CST#3972), Snail1 (ABclonal#A5243), pIGF‐1R (CST#3021), IGF‐1R (20254‐1‐AP, Proteintech), IRS‐1 (17509‐1‐AP, Proteintech), pVEGFR2 (CST#3817S), VEGFR2 (CST#9698), SHC (10054‐1‐AP, Proteintech), pAKT (CST#4060), pERK1/2 (CST#4370), pJAK1 (CST#3331), AKT (CST#9272), N‐cadherin (22018‐1‐AP, Proteintech), ERK1/2 (CST#4695), JAK1 (CST#3344), LIFR (22779‐1‐AP, Proteintech), His (CST#12698), GP130 (21175‐1‐AP, Proteintech), αSMA (55135‐1‐AP, Proteintech), E‐cadherin (20874‐1‐AP, Proteintech), Smad2/3 (CST#8685), Fibronectin (15613‐1‐AP, Proteintech), FAP (ABclonal#A6349), collagen‐I (14695‐1‐AP, Proteintech), pSmad2/3 (CST#8828), TWIST1 (25465‐1‐AP, Proteintech), and GAPDH (CST#5174) were used at recommended dilutions followed by HRP‐conjugated antibodies and analyzed with the ECL system.
9
Immunohistochemical Detection of pSTAT3
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The lung tissue slices were dewaxed, rehydrated, and subjected to immunohistochemical staining using a two‐step assay kit (PV‐9000; Zsbio Commerce Store, Inc.) according to the kit's instructions. The slices were co‐incubated with primary antibody pSTAT3 (1:200; Proteintech) at 4°C overnight, and then with an HRP‐labelled secondary antibody 37°C for 1 hour.
10
Skin Tissue Protein Extraction and Analysis
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Radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Beijing, China) containing a protease inhibitor (TransGene Biotech, Beijing, China) and a phosphatase inhibitor (TransGene Biotech) was used to extract proteins from the skin tissues or cells. The extracted proteins were separated by SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane, followed by incubation with the following primary antibodies: COLI (ProteinTech Group, Rosemont, IL), COLIII (ProteinTech Group), α-SMA (ProteinTech Group), STAT3 (ProteinTech Group), p-STAT3 (ProteinTech Group), STAP-2 (Abcam, Cambridge, MA), acidic FGF (catalog number 38145; Signalway Antibody, College Park, MD), basic FGF (catalog number 38109; Signalway Antibody), KGF-2 (catalog number 32224; Signalway Antibody), phosphorylated protein kinase B, protein kinase B, phosphorylated p38, p38, phosphorylated ERK1/2, ERK1/2, and anti-GAPDH (ProteinTech Group) (Supplementary Tables S3 andS4). The blot membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies. After development with enhanced chemiluminescence reagents, the protein bands on each membrane were quantified using Image software. The standard procedures for coimmunoprecipitation were used as previously described (Sekine et
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