The tissues and cells’ samples were solubilized in RIPA lysis at 4°C and the protein was quantified using BCA assay; 50 µg protein was separated with 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and transferred on to a polyvinylidene difluoride membrane. Membrane was blocked with 5% non-fat in TBST for 1 h at 37°C and incubated with Gal-3 (1:500, Cell Signaling Technology), TXNIP (1:500, Cell Signaling Technology), NLRP3 (1:500, Cell Signaling Technology) and GAPDH (1:2000, Cell Signaling Technology) at 4°C overnight. Membrane was washed with TBST and incubated with goat anti-rabbit monoclonal IgG (1:10000; Cell Signaling Technology) secondary antibodies at room temperature for 1 h. Membrane was visualized with an ECL kit (Pierce Chemical Co.) and analyzed by Image-Pro Plus 6.0 software.
Txnip
Manufactured by Cell Signaling Technology
Sourced in United States, China
TXNIP is a protein that functions as a regulator of cellular redox homeostasis. It binds to and inhibits the activity of thioredoxin, a key antioxidant protein.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (8 mentions)
Nlrp3, by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Cleaved caspase 1, by Cell Signaling Technology (4 mentions)
Nlrp3, by Abcam (3 mentions)
Nf κb, by Cell Signaling Technology (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Il 1β, by Cell Signaling Technology (2 mentions)
Lamin b, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Il 18, by Abcam (2 mentions)
Il 1β, by Abcam (2 mentions)
Gsdmd, by Cell Signaling Technology (2 mentions)
Sirt1, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
35 protocols using txnip
1
Immunoblotting Analysis of Gal-3, TXNIP, NLRP3 Proteins
2
Xanthohumol Protects Against Oxidative Stress
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Xanthohumol (Xn), purity >98%, was provided by the Chengdu Herbpurify CO., LTD (Chengdu, China). LPS (Escherichia coli 055:B5) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Compound C and Brusatol (specific inhibitors of the AMPK, and Nrf2, respectively), and tert-butyl hydroperoxide (t-BHP) were offered by Sigma-Aldrich (St. Louis, MO). Penicillin and streptomycin, Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were acquired from Invitrogen-Gibco (Grand Island, NY). Antibodies against Nrf2, Keap1, GCLC, HO-1, NQO1, GCLM, Trx-1, Txnip, NLRP3, casapase-1, ASC, IL-1β, Lamin B and β-actin were supplied by Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). The Annexin V fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) apoptosis kit were offered from Beyotime Institute of Biotechnology (Jinlin, China). In addition, GSH, MDA, SOD and MPO test kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were offered by Sigma-Aldrich (St. Louis, MO, USA), if not otherwise indicated.
3
Immunofluorescence Analysis of TLR4 and TXNIP
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The expressions of TLR4, TXNIP in the PC-12 cells and The expression of TLR4 in BV2 cells were evaluated by immunofluorescence. Briefly, the cells were fixed with 4% paraformaldehyde for 30 min and washed three times in PBS (5 min/time). Subsequently, the cells were punched with 0.3% Triton X100 for 15 min and blocked with 5% BSA for 2 h, The primary antibodies TLR4 (Santa cruz, sc-293072, 1:50) and TXNIP (Cell Signaling Technology, #14715, 1:50) were incubated at 4°C overnight. After washing with phosphate buffer saline (PBS), the cells were incubated with a fluorescence-conjugated antibody (1:400) for 2 h at room temperature. After washing three times with PBS (5 min/time) and then with 4',6-diamidino-2-phenylindole (DAPI) for 5 min. Cells were observed and captured with inverted fluorescent microscope (Nikon, Ts2R, Japan).
4
Whole Cell Lysate Protein Analysis in HK-2 Cells
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Whole cell lysate was prepared from cultured HK-2 cells in RIPA buffer (Biyuntian Bioengineering Institute, China). Soluble proteins from the culture medium were prepared as described previously. After measuring the protein concentration using BCA kit (Biyuntian Bioengineering Institute), equal amounts of total proteins were separated on SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The target protein was detected with one of the following primary antibodies (all from Cell Signaling, MA, USA, unless otherwise indicated) at 4 °C overnight: NLRP3 (15101S; Cell Signaling, MA, USA), TLR4 (bs-20594R; Bioss Bioengineering Institute, China), cleaved caspase 3 (9664S; Cell Signaling), caspase-3 (9665S; Cell Signaling), BAX (5023S; Cell Signaling), NF-κB (bs-0465R; Bioss Bioengineering Institute), p–NF–κB (3033T; Cell Signaling), TXNIP (14715S; Cell Signaling), and ABCG2 (bs-0662R; Bioss Bioengineering Institute). After the incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. The signal was developed using the ECL system (Biyuntian Bioengineering Institute) according to the manufacturer's instructions. The signal density was analyzed using Quantity One Software (Bio-Rad, CA, USA) and the relative protein level was calculated as the density ratio of the target protein to β-actin (4970T; Cell Signaling).
5
Western Blot Analysis of Endothelial Proteins
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Endothelial cells were lysed in modified Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium desoxycholate, 1% Triton X-100, 150 mM NaCl, 1× protease inhibitor cocktail). Proteins were separated by SDS–polyacrylamide gel electrophoresis gradient (4–20%) gel and transferred onto nitrocellulose membranes and incubated overnight at 4°C with primary antibodies. The following primary antibodies were used in this study: PLVAP (DSHB, Cat#MECA-32); PTGS1 (Cell Signaling Technologies, Cat#9896S); TXNIP (Cell Signaling, Cat#71632); FOXP1 (Cell Signaling Technologies Cat#4402S); TFRC (DSHB, Cat#G1/221/12); ZFP36L2 (Cell Signaling, Cat#2119); ACTN1 (Sigma, Cat#A5044); GAPDH (Millipore Sigma, Cat#MAB374); VCL (Millipore Sigma, Cat#V-9131). Secondary antibodies included: Amersham ECL Rabbit IgG HRP-Linked Whole Antibody (Cat#NA934) and Amersham ECL Mouse IgG, HRP-Linked Whole Antibody (Cat#NA931) both from Cytiva. Immuno-complexes were detected by enhanced chemiluminescence with SuperSignal West Pico PLUS (Thermo Fisher Scientific #PI34580) and Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific #PI34096) using ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Quantification of bands by densitometry analysis was performed using ImageLab Software (Bio-Rad Laboratories).
6
Pullulan-based Fluorescent Probe Synthesis
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Pullulan was purchased from the Tokyo Chemical Industry (Tokyo, Japan). Isopropyl alcohol, citric acid, fluorescein isothiocyanate isomer I (FITC), sodium hydroxide (NaOH, 99.0%), monochloroacetic acid (ClCH2COOH), lithium aluminum hydride (LiAlH4), phosphate buffered saline (PBS, pH 7.4), and dialysis tubing cellulose membranes (Mw. cutoff = 14 K), (±)-verapamil hydrochloride, fatty acid-free bovine serum albumin (BSA), and palmitate were purchased from Sigma Aldrich (St. Louis, MO, USA). Glycerol, methanol, and ethanol were purchased from Duksan Chemicals (Seoul, South Korea). Immunoblotting and immunostaining were performed using antibodies against p62, NLRP3, cleaved caspase-3, phospho-CaMKII, TXNIP (Cell Signaling Technology, Danvers, MA, USA), p62 (Sigma-Aldrich), ubiquitin, CaMKII, caspase-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-tubulin, β-actin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and GAPDH (Aviva Systems Biology, San Diego, CA, USA). Triple-distilled and deionized water were used throughout the experiment.
7
Immunoprecipitation of TXNIP and NLRP3
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Referring to prior research [40 (link)], cells (N = 1 × 107) from each group were lysed with a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerinum, 1 mM ethylene damine tetraacetic acid, 0.5% NP – 40 and protease inhibitor), and the cell debris was removed by means of centrifugation. After the concentration of lysis solution was determined with a BCA reagent, equal amounts of protein were collected from each group and supplemented with the same volume of cell lysis solution. Subsequently, each sample was incubated with the following antibodies: TXNIP (# 14715S, dilution ratio of 1:50, Cell Signaling Technology), NLRP3 (ab263899, dilution ratio of 1: 30, Abcam), and 15 μL protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. After 3 rounds of washing, the beads were harvested using centrifugation, added with an equal volume of reducing loading buffer, and boiled at 100°C for 5 min. Afterward, the samples were isolated with SDS-PAGE and transferred onto PVDF membranes (Millipore, Temecula, CA, USA) for Western blot analysis.
8
Salidroside Inhibits Neuroinflammation in PD
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Salidroside was provided by the Second Medical University (Shanghai, China; purity > 99%). MPTP-HCl was purchased from MedChemExpress (New Jersey, USA). Lipopolysaccharide (LPS) was purchased from Sigma Aldrich (St. Louis, USA). Enzyme-linked immunosorbent assay (ELISA) kits of interleukin (IL)-18 and IL-1β were purchased from Elabscience (Wuhan, China). The primary antibodies against MyD88, p-IκBα, IκBα, NF-κB, ASC, cleaved-Caspase-1, GSDMD, α-Synuclein, TH, TXNIP and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-p-NF-κB, TXNIP, NLRP3, IL-1β and IL-18 primary antibodies were produced by Abcam (Cambridge, UK). The anti-TLR4 primary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies are listed in Supplementary Table 1 and the critical chemicals are listed in Supplementary Table 2 .
9
Carnosic Acid Regulates Cell Apoptosis
10
Rosiglitazone Ameliorates Diabetic Nephropathy
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Rosiglitazone (ROG, 1 mg/pill) was purchased from the Chengdu Hengrui Pharmaceutical Co. (Chengdu, People’s Republic of China). Alloxan was purchased from the Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Commercial reagent kits, including BUN, SCr, UUA, UCr, urine protein, TC, TG, TNF-α, and IL-6, were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, People’s Republic of China). All chemical reagents used in the present study were purchased from Nanjing Chemical Reagent Co., Ltd (Nanjing, People’s Republic of China). Primary antibodies against phospho-NF-κBp65, NF-κBp65, phospho-IκB, IκB, TXNIP, phospho-mTOR, mTOR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA).
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