ATDC5 cells were treated with 5 µg/ml LPS for 5 h after 48 h of transfection. Cells were washed with ice-cold PBS and then lysed with RIPA (Beyotime Institute of Biotechnology) buffer with 1% PMSF at 4°C for 1 h. Protein samples were collected by centrifugation for 5 min at 4°C at 12,000 × g. Protein concentration was determined using a bicinchoninic acid protein assay kit. Proteins (30 µg/lane) were separated by SDS-PAGE on 10% gels, electroblotted onto PVDF membranes and then blocked in 5% non-fat milk at room temperature for 2 h. Membranes were then incubated with primary antibodies: IL-6R (cat. no. ab83053; 1:1,000; Abcam), STAT3 (cat. no. ab119352; 1:1,000; Abcam), phosphorylated STAT3 (cat. no. ab76315; 1:1,000; Abcam) and GAPDH (cat. no. ab181602; 1:1,000; Abcam), overnight at 4°C and washed with PBS-0.1% Tween-20 (PBST) four times. Subsequently, the membranes were incubated with the HRP-conjugated anti-rabbit secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) for 2 h at room temperature, the membranes were then washed with PBST four times. Finally, ECL reagent (EMD Millipore) was used to visualize the protein bands using a FluorChem FC3 system (ProteinSimple). AlphaView 3.4.0 software (ProteinSimple) was used for the quantification of the protein bands. The experiments were repeated three times.
Phosphorylated stat3
Manufactured by Abcam
Sourced in United Kingdom, United States
Phosphorylated STAT3 is a recombinant protein that represents the phosphorylated form of the Signal Transducer and Activator of Transcription 3 (STAT3) protein. STAT3 is a transcription factor that plays a crucial role in various cellular processes, including cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Abcam (4 mentions)
Phosphorylated akt, by Abcam (2 mentions)
Ecl reagent, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Phosphorylated iκb α, by Abcam (1 mentions)
Anti mouse igg1 hrp, by Abcam (1 mentions)
Cathepsin k, by Abcam (1 mentions)
Mcp 1, by Abcam (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Stat1, by Abcam (1 mentions)
Phosphorylated stat1, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cxcl12, by Cell Signaling Technology (1 mentions)
α sma, by Abcam (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Total akt, by Abcam (1 mentions)
10 protocols using phosphorylated stat3
1
LPS-induced IL-6R and STAT3 Activation Assay
2
Flemingia philippinensis Anti-Inflammatory Assay
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The Flemingia philippinensis root was purchased from a traditional herb market in Guilin, China, and authenticated by a taxonomist. The voucher specimen was stored in Pharmacy College, Guilin Medical University, Guilin, China. Chicken type collagen (CII) and Complete Freund's Adjuvant were purchased from Sigma-Aldrich. Antibodies to the following proteins were purchased from Santa Cruz Biotechnology, Santa Cruz, CA: goat anti-mouse IgG2a-HRP (sc-2061), MMP-9 (sc-393859), cathepsin K (sc-48353), MCP-1 (sc-52701), phosphorylated STAT3 (sc-8059), phosphorylated I-κBα (sc-8404), and phosphorylated NF-κBp65 (sc-101753); antibodies anti-Mouse IgG1-HRP, phosphorylated JNK1+JNK2+JNK3 (ab124956), phosphorylated p38 MAPK (ab178867), and phosphorylated Jun D+c-Jun (ab208035) were purchased from Abcam; phosphorylated ERK1/2 antibody (4370) was from Cell Signaling Technology, GAPDH antibody was from PeproTech Biotechnology, and ELISA kits for TNF-α, IL-10, IL-12/IL-23 p40 (total), and MCP-1 were from eBioscience.
3
Immunohistochemical Analysis of Tumor Samples
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Tumor tissues from patients were kept in 4% PFA overnight, then processed, embedded in paraffin, and sectioned at 4 μm for further study. Sections of tumor tissues from patients were blocked with blocking solution containing 5% bovine serum albumin for 30 min and stained with primary antibodies AhR (1:100; Abcam, Cambridge, UK), phosphorylated AKT (1:300, Abcam, Cambridge, UK) and phosphorylated STAT3 (1:500 Abcam, Cambridge, UK) at 4°C overnight, followed with incubation with secondary antibodies (1:1000; Abcam, Cambridge, UK) for 1 hour at room temperature. Nuclei were stained with the DAPI solution (1 µg/ml). Confocal microscope (Olympus, Tokyo, Japan) was used to visualize the sections. The fluorescence intensity of section and relative protein expression was analyzed by image J software 1.8.0 (NIH, Bethesda, Maryland, USA).
4
Western Blot Analysis of Phosphorylated STAT Proteins
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The tumor tissue homogenate solution was lysed by lysis buffer containing protease inhibitors to extract the total proteins. Then, the samples were loaded onto 10% SDS-PAGE gels of separation and transferred to the PVDF membrane (Millipore Sigma, St. Louis, MO, USA). Bovine serum albumin (5%) in Tris-buffer saline containing 0.1% Tween-20 (TBST) was used to block the unspecific proteins for 1 h at room temperature. The primary antibodies were used by diluting 1000 times with TBST buffer. After incubating with the following primary antibodies: phosphorylated Stat1 (Abcam, Cambridge, UK), Stat1 (Abcam, Cambridge, UK), phosphorylated Stat3 (Abcam, Cambridge, UK), or Stat3 (Abcam, Cambridge, UK). Rabbit anti-β-actin (Perprotech, China) was employed as a loading control, the membranes were incubated with HRP-labeled secondary antibodies for 1 h at room temperature. The secondary antibody was used by diluting 2000 times with TBST buffer. Finally, protein expression was quantitatively measured and visualized by SuperLumia ECL HRP Substrate Kit (Servicebio) and Image J.
5
Molecular Mechanism of CXCL12 Signaling
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Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) at different time points. The L4/6 DRG was immediately removed and homogenized on ice in 15 mmol/l Tris containing a cocktail of proteinase inhibitors and phosphatase inhibitors. Protein samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The blots were placed in the blocking buffer for 1 h at room temperature and incubated with primary antibodies against CXCL12 (1:1000; Cell Signaling Technology, USA), phosphorylated STAT3 (1:1000, Abcam, USA), STAT3 (1:1000, Abcam, USA), or β-actin (1:2000, Cell Signaling Technology, USA) overnight at 4℃. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody. ECL (Pierce, USA) was used to detect the immune complex. The band was quantified with a computer-assisted imaging analysis system (NIH ImageJ).
6
Protein Expression Analysis of A498 and Renca Cells
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The A498 cells and Renca cells were collected for protein samples with NP40 solution. 25 μg protein samples were separated by SDS-PAGE, followed with transferring to PVDF membranes. Next, samples were examined by immunoblotting with primary antibodies against: α-SMA (1:1000, Abcam, Cambridge, UK), AhR (1:5000, Abcam, Cambridge, UK), phosphorylated-Src (1:500, Abcam, Cambridge, UK), total Src (1:500, Abcam, Cambridge, UK), phosphorylated AKT (1:500, Abcam, Cambridge, UK), total AKT (1:500, Abcam, Cambridge, UK), phosphorylated STAT3 (1:500 Abcam, Cambridge, UK), total STAT3 (Abcam, Cambridge, UK) and β-actin (1:1000, Abcam, Cambridge, UK) respectively at 4°C overnight. Then HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) was incubated for 1 hour at room temperature, and visualized by using ECL detection kit (Thermo, MA, USA).
7
Western Blotting for Protein Expression
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An amount of 25 µg of protein was separated by polyacrylamide gel and then electro-transferred to polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) for immunoblotting analysis. The LGALS9 antibody (1:600, Abcam, Cambridge, UK), phosphorylated STAT3 (1:200, Abcam), and GAPDH murine antibody (1:800, Abcam) were respectively used to incubate with the membrane at 4°C overnight. After incubation with the appropriate HRP-conjugate secondary antibody, the abundance of the proteins was detected using a ChemiDoc XRS Imaging System (Bio-Rad Laboratories Inc.).
8
Immunohistochemical Analysis of Spinal Cord Tissues
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Immunohistochemistry was performed as previously described [22 (link)]. Briefly, animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and cardiac perfusion were performed using 0.9% physiological saline, followed by 4% paraformaldehyde in PBS. Next, L4–L6 spinal cord tissues were removed and postfixed in the same fixative overnight and then dehydrated with 30% sucrose. Cryostat sections (16 μm thick) were cut and processed for immunofluorescent staining with primary antibodies for SIRT1 (1:400, Santa Cruz, USA), NALP1 (1:50, Novus Biologicals, USA), phosphorylated STAT3 (1:100, Abcam, UK), NeuN (1:500, Chemicon, USA), GFAP (1:800, Chemicon, USA), or OX42 (1:250, Chemicon, USA). After incubation overnight at 4 °C, the sections were then incubated with cy3-conjugated and fluorescein isothiocyanate-conjugated secondary antibodies for 1 h at room temperature. Isotype IgG was applied in separated sections as control, which did not yield detectable immunosignal. The stained sections were then examined with a Leica (Leica, Germany) fluorescence microscope, and images were captured with a Leica DFC350 FX camera.
9
Sodium Pentobarbital-Induced Neuronal Protein Profiling
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The L4 to L6 DRGs were immediately removed at different time points after application of sodium pentobarbital at 50 mg/kg dose (i.p.) and were homogenized on ice in 15 mM tris buffer containing the inhibitors of proteinase (Roche) and phosphatase (Roche) on ice. Protein samples were separated by SDS-PAGE and then transferred onto a PVDF membrane. The PVDF membrane was incubated with primary antibodies against Nav1.6 (1:200; Alomone labs), TNF-α (1:500; Santa Cruz), phosphorylated STAT3 (1:1000; Abcam), STAT3 (1:1000; Abcam), acetylated histone H3 (K9) (1:1000; Abcam), acetylated histone H4 (Ac-H4, 1:1000; Millipore), p300 (1:1000; Abcam), or β-actin (1:1500; CST), after blocking with the block buffer for 1 h at room temperature (RT). Enhanced chemiluminescence (ECL) (Pierce) was used to detect the immunocomplex after the blots were incubated with the secondary antibodies. The intensity of the band was examined by a computer-assisted imaging analysis system (ImageJ, NIH).
10
Comprehensive Protein Expression Analysis
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Standard western blot was performed using whole-cell protein lysates, and primary antibodies BATF2, MAT2A, FOXM1(Abcam),phosphorylated STAT3(p-STAT3), phosphorylated STAT1(p-STAT1), phosphorylated p65 (p-p65),Bcl-2, BAX, caspase-3, caspase-9 and GAPDH (bioswamp, China), a secondary antibody (anti-rabbit IgG ;bioswamp, China). Equal protein-sample loading was monitored using an anti-GAPDH antibody.
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