The total proteins from tissue and cell samples were washed three times with PBS. Then the samples were lysed in RIPA lysis buffer for 30 min. The protein lysates were separated by 10% SDS-PAGE. The specific proteins were transferred on PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 30 min and then washed twice with TBST. The specific proteins were incubated with the different primary antibodies overnight at 4°C. The used primary antibodies were as follows: JAK1 (Ab-1022), p-JAK1 (SAB4300123), JAK2 (Ab-570), p-JAK2 (SAB4301238), p-STAT3 (SAB4300034), Ki67 (SAB4501880) and STAT3 (SAB4502871) (Sigma-Aldrich), cleaved caspase-3 (9654), matrix metalloproteinases [MMP-2 (40994) and MMP-9 (15561)], E-cadherin (14472), N-cadherin (4068), Snail (3879) and β-actin (8457) (Cell Signaling Technology, Beverly, MA, USA). The membranes were washed twice and then incubated with HRP-linked secondary antibody (7075; Cell Signaling Technology) for 1–2 h. Protein signals were visualized using enhanced chemiluminescence reagents (ECL; GE Healthcare, Waukesha, WI, USA).
Pstat3
Manufactured by Merck Group
Sourced in United States
The PStat3 is a precision potentiostat, a type of electrochemical instrumentation used for a variety of analytical and research applications. It provides precise control and measurement of electrochemical parameters, enabling accurate and reliable data collection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
β actin, by Merck Group (3 mentions)
Stat3, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Nf kb, by Cell Signaling Technology (2 mentions)
Pnfkb, by Cell Signaling Technology (2 mentions)
P atf2, by Cell Signaling Technology (2 mentions)
Pc jun, by Merck Group (2 mentions)
Patf 2, by Merck Group (2 mentions)
Protease inhibitor and phosphatase inhibitor cocktail 2 3, by Merck Group (2 mentions)
Pnfkb, by Merck Group (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
P c jun, by Cell Signaling Technology (2 mentions)
Sb202190, by Merck Group (2 mentions)
Stat3, by BD (2 mentions)
Rrgfr α1, by R&D Systems (2 mentions)
Rmgfr α2, by R&D Systems (2 mentions)
Rrgfr α3, by R&D Systems (2 mentions)
Rrgndf, by R&D Systems (2 mentions)
14 protocols using pstat3
1
Western Blot Analysis of Protein Signaling
2
Western Blot Analysis of Metabolic Enzymes
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For Western analysis antibodies to AKR1C2 (H00001646-D01, Abnova), AKR1C3 (H00008644-B01, Abnova), AKR1C4 (H00001109-M01, Novus), AKT1 (#2967, Cell Signaling), Caspase3 (sc-7148, Santa Cruz), SLC16A3 (OAAB08662, Aviva Systems Biology) PARP-1 (sc-74470, Santa Cruz), STAT3 (sc-81385, Santa Cruz), pSTAT3 (S2690, Sigma) and β-actin (ab8226, Abcam) primary antibodies and HRP-conjugated secondary antibodies (Dako) were used. Melphalan, ursodeoxyholate, indomethacin, flufenamic acid, dichloroacetic acid, 2-deoxy-D-glucose, sodium oxamate, metformin, oligomycin, antimycinA, FLLL31, wortmannin, rapamycin, methyl glyoxal, acetylsalicylic acid, ibuprofen, (Sigma Aldrich), tert-butyl peroxide (Fluka), LY294002, SB203580 and BIRB0796 (Cell Signaling) were used in viability assays.
3
Investigating Transcription Factor Activation
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LECs (2×106) treated with Stattic, SP600125, IL6 or EGM, were used to prepare cell lysates or nuclear extracts. Five hundred microliters of cell lysates or 200 µL nuclear extracts were incubated overnight at 4°C with antibodies suitable for IP (1:100 diluted): pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Cell Signaling). Ten microliters of Protein A/G Plus Agarose (Santa Cruz Biotech) was added and incubated for 3 h at 4°C. The beads were rinsed 3 times with 500 µL cell lysis buffer for IP (Pierce) supplemented with the protease inhibitor and phosphatase inhibitor cocktail 2/3 (Sigma). The protein complex was reduced and separated by SDS-PAGE and probed with the following antibodies in a Western assay: pStat3, pc-Jun, pATF-2, pNFkB, and NFkB (Sigma). All the original gel images of immunoblot analyses are presented in Supplementary Fig. 25 .
4
Toxoplasma gondii Infection Immunostaining
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For immunostaining study, AHNP (S-NSC) cells were grown on glass cover slip before infected with Toxoplasma gondii. 24 hours after infection, cells were fixed with paraformaldehyde and standard immunefluroescent staining protocol was adopted for studying the localization of targets of interest, including NFκB, STAT3, tyrosine hydroxylase, and dopamine. Antibodies utilized include: anti-NFκB p50 (Santa Cruz Biotech, SC-7178); P-STAT3 (Y705), cell signaling (#91315); anti-tyrosin hydroxylase (Sigma, T1299); and dopamine antibody (Novus Biologicals, NB120-1001).
5
Regulation of ILC3 cell signaling pathways
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Sorted intestinal ILC3 cells were starved for 3 hours in RPMI at 37°C in order to ensure ILC3 viability. Retfl or RetΔ were analysed directly ex vivo. To test ERK, AKT, p38-MAPK (Cell Signaling Technology) and STAT3 (BD Pharmigen) upon GFL stimulation WT ILC3 were activated with 500ng/mL (each GFL) and co-receptors (rrGFR-α1, rmGFR-α2, rrGFR-α3 and rrGNDF from R&D Systems; rhNRTN and rhARTN from PeproTech) for 10 and 30min. When referring to the use of ‘GFL’, we have employed GDNF, NRTN, ARTN and their specific co-receptors in combination. For inhibition experiments cells were incubated 1h at 37°C before GFL stimulation, to test ERK, AKT, p38/MAPK and STAT3 phosphorylation, or during overnight stimulation with GFLs, to determine Il22 expression levels. Inhibitors were purchased from Sigma-Aldrich: p38 MAPK/ERK-AKT - LY294002 (LY); ERK - PD98059 (PD); AKT - AKT Inhibitor VIII (VIII); p38 MAPK - SB 202190 (SB); and pSTAT3 – S3I-201 (S3I).
6
Comprehensive Protein Analysis Protocol
7
Protein Expression Analysis in ASMCs
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Total protein was extracted from ASMCs using RIPA lysis buffer (Fermentas, Glen Burnie, MD, U.S.A.). After determination of protein concentration, equal amount of protein extracts was separated by 10–12% SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A.). After incubation with blocking buffer, 5% non-fat milk, for 1 h, primary antibodies against α-SMA (1:500; Abcam, Cambridge, MA, U.S.A.), myocardin (1:500; Sigma–Aldrich, St. Louis, MO, U.S.A.), nuclear factor-κ B (NF-κB) p65 (1:500; Sigma–Aldrich), p-p65 (1:500; Abcam), signal transducer and activator of transcription 3 (STAT3; 1:500; Sigma–Aldrich), p-STAT3 (1:500; Sigma–Aldrich), p-AKT (1:1500; Abcam), AKT (1:2000; Abcam), and β-actin (1:500; Abcam) were added to the incubation system and incubated at 4°C overnight. Then the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000; Abcam) at 37°C for 1 h. The protein bands on the membranes were visualized using ECL reagent (ECL, Pierce, Rockford, IL, U.S.A.). The protein levels were analyzed by detecting the density using ImageJ Software (National Institutes of Health, NIH, Bethesda, MD, U.S.A.).
8
Investigating Transcription Factor Activation
9
Assessing Compound Effects on Cellular Proteins
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To assess the effects of compounds on IκBα protein expression, cells were treated with the substances for 30 min before exposure to LPS (1 μg/mL) for one hour.40 (link) Also, to investigate the expression of STAT3, p-STAT3, BAX, and Bcl-2 proteins, cells received IL-6 (50 ng/mL) for four hours after treatment with compounds.41 (link) The supernatant of centrifuged lysates was used for western blot analysis. The protein content was evaluated using a BCA test kit (Thermo Fisher Scientific, Oxford, UK). The samples were electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, St. Louis Missouri, USA) after loading on 10% SDS-PAGE gels. After membrane blocking, the membranes were coated with the primary antibodies at 1:1000 dilution against BAX, Bcl-2, IκBα, STAT3, p-STAT3, and β-actin (Sigma-Aldrich, Munich, Germany) overnight at 4 °C. Afterward, the membranes were then coated with HRP-conjugated anti-mouse immunoglobulin G secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, California, USA). Eventually, enhanced chemiluminescence (ECL) (SuperSignal, Thermo Fisher Scientific, Oxford, UK) was used for detection.
10
Regulation of ILC3 cell signaling pathways
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