Cells were cultured for 48 hours on glass coverslips in 6-well plate and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature, incubation with primary antibody for one hour, wash with 1% Triton X-100, and incubation with Alexa Fluor-conjugated secondary antibodies for one hour in the dark. The coverslips were stained with Hoechst 33342 (H3570, Invitrogen, 1:3000) before being mounted with VECTASHIELD mounting medium (H-1000, Vector laboratories). Images were captured by Leica TCS-NT 4D confocal microscope. Z stacks were collected with a spacing of 1 μm. Antibody and dilutions used in the studies: anti-Flag (14793S, Cell signaling, 1:100), anti-Lamin B1 (68591S, Cell signaling, 1:100), anti-STAT3 (MA1–13042, Thermo Fisher Scientific, 1:100). Alexa Fluor 488 Goat anti-Rabbit (A11008, Life technologies, 1:5000), Alexa Fluor 594 Goat anti-Mouse (A11032, Life technologies, 1:5000). The fluorescence-intensity profile along the Z-axis from confocal Z-stacks were shown. The fluorescence intensity of nuclear localized STAT3 was quantified using the confocal software to define the selected ROI (Region of Interest) area based on nuclear DAPI signal. More than 200 cells were quantified in at least three independent experiments.
Anti stat3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-STAT3 is a laboratory reagent that detects and quantifies the expression levels of the STAT3 protein. STAT3 is a transcription factor involved in cellular processes such as cell growth and survival. The Anti-STAT3 product can be used in various research applications to study the role of STAT3 in different biological systems.
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Lab products found in correlation
Anti p stat3, by Thermo Fisher Scientific (3 mentions)
Anti jak2, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Tcs nt 4d confocal microscope, by Leica (2 mentions)
Ma1 13042, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Cell Signaling Technology (2 mentions)
Anti lamin b1, by Cell Signaling Technology (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Biophotometer plus, by Eppendorf (2 mentions)
Anti stat1, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Thermo Fisher Scientific (2 mentions)
Biorad protein dye, by Bio-Rad (2 mentions)
Anti p stat1, by Thermo Fisher Scientific (2 mentions)
Anti stat5, by Thermo Fisher Scientific (2 mentions)
Anti p stat5, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Trichloroacetic acid, by Merck Group (2 mentions)
12 protocols using anti stat3
1
Quantitative Analysis of Nuclear STAT3 Localization
2
STAT Phosphorylation in Leukemia Cell Lines
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Protein from MV4-11, MV4-11R-cep, and MV4-11R-cep + 5-Aza cells was extracted by RIPA buffer (Sigma-Aldrich, MO, USA). The three cell lines were incubated with 300 nM CEP-701 for 3 days before protein extraction. BioRad protein dye (BioRad, Hercules, California, USA) and a spectrophotometer (BioPhotometer Plus, Eppendorf, Germany) were employed for the measurement of protein concentrations. Preparation of immunoblotting was performed as described previously [30 (link)]. Antibodies used were anti-STAT1, anti-p-STAT1, anti-STAT3, anti-p-STAT3, anti-STAT5, anti-p-STAT5, and anti-β-actin (Thermo Scientific, Waltham, MA, USA).
3
Quantifying Protein Levels in Imatinib-Treated Leukemia Cells
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Total protein was extracted from treated K562, K562-R and K562-R+5-Aza cells with 400 nM imatinib for 3 days, using RIPA buffer (Sigma-Aldrich, MO, USA). BioRad protein dye (BioRad, Hercules, California, USA) and spectrophotometer (BioPhotometer Plus, Eppendorf, Germany) were used for measurement of protein concentrations. Preparation of immunoblotting was performed as described previously (Frohling et al., 2007). Antibodies used were anti-STAT1, anti-p-STAT1, anti- STAT3, anti-p-STAT3, anti-STAT5, anti-p-STAT5 and anti-β-actin (Thermo Scientific, Waltham, MA, USA).
4
Evaluation of Pinosylvin and Resveratrol in Inflammatory Pathways
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Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum (FBS), L-glutamine, penicillin/streptomycin, phosphate buffered saline (PBS), bovine serum albumin (BSA), protease inhibitors, trypan blue, lipopolysaccharide from E. coli (LPS), trichloroacetic acid (TCA), sulphorhodamine B (SRB), Tris base (Tris[hydroxymethyl]aminomethane), pinosylvin (HPLC, ≥97%), and resveratrol were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). ECL System was from Bio-Rad. RAW 264.7 cells were purchased from ATCC, Glasgow, UK (No. TIB-71), employed Abs anti-phosphoJak2 (#PA538287), anti-Jak2 (#PA511267), anti-phosphoStat3 (#PA5121259), and anti-Stat3(#PA5120138) were from ThermoFisher Scientific. Anti-β-actin (AC-15; sc-69879) was from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. Water and acetonitrile were HPLC-grade, while all other solvents were reagent-grade, obtained by VWR International s.r.l. (Milan, Italy).
5
Antibody-Based Protein Analysis Protocol
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Antibodies employed in this study were as follows: anti‐STAT3 phosphorylated on tyrosine 705 (P‐STAT3 Y705), anti‐STAT3 phosphorylated on Serine 727 (P‐STAT3 S727), and anti‐STAT3 from Thermo Fisher Scientific; anti‐γ‐H2AX, rabbit polyclonal (4937; Cell Signaling); anti‐p21, rabbit polyclonal (MA5‐14949; Invitrogen); anti‐Emerin, mouse monoclonal (MONX10804; Monosan); anti‐β‐tubulin; anti‐lamin A/C, goat polyclonal (SC‐6215; Santa Cruz Biotechnology); anti‐Progerin, mouse monoclonal (13A4; Enzo); anti‐GAPDH, mouse monoclonal (MAB374; Millipore); anti‐Ankrd2, mouse monoclonal, clone YAS11 (LS‐Bio); and anti‐collagen VI, monoclonal (Millipore).
6
Quantitative Analysis of Nuclear STAT3 Localization
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Cells were cultured for 48 hours on glass coverslips in 6-well plate and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature, incubation with primary antibody for one hour, wash with 1% Triton X-100, and incubation with Alexa Fluor-conjugated secondary antibodies for one hour in the dark. The coverslips were stained with Hoechst 33342 (H3570, Invitrogen, 1:3000) before being mounted with VECTASHIELD mounting medium (H-1000, Vector laboratories). Images were captured by Leica TCS-NT 4D confocal microscope. Z stacks were collected with a spacing of 1 μm. Antibody and dilutions used in the studies: anti-Flag (14793S, Cell signaling, 1:100), anti-Lamin B1 (68591S, Cell signaling, 1:100), anti-STAT3 (MA1–13042, Thermo Fisher Scientific, 1:100). Alexa Fluor 488 Goat anti-Rabbit (A11008, Life technologies, 1:5000), Alexa Fluor 594 Goat anti-Mouse (A11032, Life technologies, 1:5000). The fluorescence-intensity profile along the Z-axis from confocal Z-stacks were shown. The fluorescence intensity of nuclear localized STAT3 was quantified using the confocal software to define the selected ROI (Region of Interest) area based on nuclear DAPI signal. More than 200 cells were quantified in at least three independent experiments.
7
Western Blot Analysis of Protein Expression
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The cells transfected for 48 hours were cleaved using RIRP Lysis Buffer (Thermo Fisher Scientific). Then, the supernatant was collected after centrifugation 4 times at 14 000 rpm for 15 minutes. The protein concentration was detected by bicinchoninic acid (BCA) kit (Bio-Rad, USA). The target protein was separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred into PVDF (polyvinylidene difluoride) membrane overnight. After the blots were sealed using 10% skim milk, the primary antibody was used to hybridize with the target protein overnight at 4°C (anti-MRP1, 1: 1000; anti-GST-π, 1: 2000; anti-ABCB1, 1: 1000; anti-GAPDH, 1: 5000; anti-SOCS2, 1: 1000; anti-SOCS4, 1: 1000; anti-SOCS5, 1: 1000; anti-p-JAK2, 1: 2000; anti-JAK2, 1: 2000; anti-p-STAT3, 1: 2000; anti-STAT3, 1: 2000; Thermo Fisher Scientific). Subsequently, the blots were incubated with horseradish peroxidase conjugated secondary antibody (1: 10 000) after washing in TBST. We used an ECL (enhanced chemiluminescence) kit (Abcam, USA) developed the blots.
8
Intestinal Protein Analysis Protocol
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Proteins were isolated from jejunum samples and analyzed as described previously (Xie et al., 2020 (link)). Total protein was extracted from the intestinal tissues using radioimmunoprecipitation assay lysis buffer, and the protein concentration in the homogenates was determined using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, Illinois, USA). The proteins and color pre-stained protein marker (M222-10, GenStar, Beijing, China) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts, USA). The band densities were quantified using ImageJ software (version 1.8.0, National Institute of Health, Bethesda, MD, USA). The results were presented as the ratios of target protein level to β-actin level or the total protein level. The following antibodies were used: anti-p-JAK2 (#3771, CST, Cell Signaling Technology, Beverly, MA, USA), anti-JAK2 (#3230, CST), anti-p-STAT3 (#SC-8059, Santa Cruz Biotechnology, Dallas, TX, USA), anti-STAT3 (#SC-8019), anti-ZO-1 (#339100, Thermo Fisher), anti-Claudin-1 (#374900, Thermo Fisher), and anti-β-actin (#250132, ZENBIO, Chengdu, Sichuan, China).
9
Evaluation of Anti-inflammatory Effects
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Folin-Ciocalteu reagent, aluminum chloride, chlorogenic acid, quercetin, bergapten, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), bovine serum albumin (BSA), protease inhibitors, trypan blue, phosphate buffered saline (PBS), LPS, trichloroacetic acid (TCA), sulphorhodamine B (SRB) were purchased by Sigma-Aldrich S.p.a. (Milan, Italy). ECL System was from Bio-Rad. RAW 264.7 cells were purchased by ATCC, UK (No. TIB-71). The Abs employed were anti-phosphoJak2 (#PA538287), anti-Jak2 (#PA511267), anti-phosphoStat3 (#PA5121259), anti-Stat3(#PA5120138), (all from ThermoFisher Scientific, Waltham, MA, USA), anti-β-actin (AC-15; sc-69879) from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. All other reagents were obtained by VWR International s.r.l. (Milan, Italy).
10
Molecular Signaling Pathways in Stem Cells
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Recombinant human GDF15 (R&D Systems, Minneapolis, MN, USA), imiquimod (IMQ, InvivoGen, San Diego, CA, USA), neutralizing antibody against LIF (R&D Systems), and ERK1/2 inhibitor PD98059 (Cell Signaling Technology, Danvers, MA, USA) were used in this study. The antibodies used were as follows: anti-CD133/2 (Miltenyi Biotec, Auburn, CA, USA), anti-PROM1 (Sigma Aldrich, St. Louis, MO, USA), anti-Nestin (Merck-Millipore, Billerica, MA, USA), anti-Toll-like receptor 7 (anti-TLR7, Novus Biological Inc., Littleton, CO, USA), anti-Musashi (Merck-Millipore), anti-phospho-STAT3 (p-Tyr705, Cell Signaling Technology), anti-STAT3 (Thermo Fisher Scientific), anti-SOX2 (R&D Systems), anti-ALDH1 (Thermo Fisher Scientific), anti-β-actin (TransGen Biotech, Beijing, China), anti-GDF15 (Thermo Fisher Scientific), anti-LIF (Merck-Millipore), and anti-Fos (GeneTex, Irvine, CA, USA). The Smad2/3 Antibody Sampler Kit (Cell Signaling Technology) and Smad1/5/9 Antibody Sampler Kit (Cell Signaling Technology) were also employed.
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