For production of lentiviral particles, HEK293T cells, pretreated with 25 μm chloroquine for 1 h, were transfected with vectors containing the shRNA against TXNIP (Open Biosystems, Heidelberg) and a plasmid mixture for gag, pol, env and VSV‐G for pseudotyping. 8 h post transfection medium was replaced from packaging cells. After 2 days, the supernatant was passed through a 0.45 μm filter, supplemented with Polybrene (8 μg·mL−1). 1x105 target cells were infected by spin occulation with 1 mL of viral supernatant. Stably transduced Jurkat cells were selected by puromycin (1 μg·mL−1) and Doxycycline (Dox)‐dependent shRNA expression was checked by Western blot analysis.
Txnip
Manufactured by Thermo Fisher Scientific
Sourced in United States
TXNIP is a protein that regulates the activity of thioredoxin, an important antioxidant enzyme. It plays a role in cellular metabolic processes and stress response.
Automatically generated - may contain errors
Lab products found in correlation
Cyp d, by Thermo Fisher Scientific (2 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Hpa001562, by Merck Group (1 mentions)
Axiovision z1, by Zeiss (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Trans blot turbotm transfer pack, by Bio-Rad (1 mentions)
Magt1, by Abcam (1 mentions)
Trpm7, by Fortis Life Sciences (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Claritytm western ecl substrate, by Bio-Rad (1 mentions)
Ps129 α synuclein, by Abcam (1 mentions)
β actin, by AbFrontier (1 mentions)
Chemidoc trs, by Bio-Rad (1 mentions)
Phospho p38, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
6 protocols using txnip
1
Lentiviral Transduction of Jurkat Cells
2
Lentiviral Transduction of Jurkat Cells
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CCs cells were pretreated with 25 μM chloroquine for one hour to produce lentiviral particles, transfected with a shRNA which is a vector containing against TXNIP (Open Biosystems, Heidelberg) and a plasmid mixture of env, gag, VSV-G, and pol, with in pseudotyping. Eight hours after transfection, the medium was changed. After two days, It was passed the supernatant through a 0.45 μm filter supplemented with polybrene (8 μg/ml). 1 ml of viral supernatant was used to infect with 1x10 5 target cells by spin bite method. Stably transduced Jurkat cells were selected by puromycin (1 μg/ml).
3
Immunostaining of Human and Mouse Corneas
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Three separate human corneas, ages 27, 42, and 62, were used with postmortem delay of no more than 72 hours (Eversight Eye Bank, Chicago, IL, USA). Frozen sections (5 μm) of optimal cutting temperature compound-embedded human and mouse corneas were fixed in 4% paraformaldehyde, blocked in 10% goat serum in PBS and incubated overnight with the following primary antibodies: rabbit polyclonal antibodies against Kl67 (SP6- 1:500 dil; Sigma-Aldrich Corp., St. Louis, MO, USA), PBK (16110–1-AP-1:100dil; Proteintech, Rosemont, IL, USA), TXNIP (40–3700- 1:100 dil; Thermo Scientific, Waltham, MA, USA), H2AX (10856–1-AP- 1:50 dil; Proteintech), ATF3 (HPA001562–1:50 dil; Sigma-Aldrich Corp.) or a mouse monoclonal antibody against K15 (MA5–11244–1:100 dil; Thermo Scientific). Appropriate secondary antibodies conjugated to Alexa-555 or Alexa-488 (Invitrogen, Carlsbad, CA, USA) at 1:300 dilution were used to detect primary antibodies. DAPI (4′,6-diamidino-2-phenylindole) was used to label nuclei, and mounted using Gelvatol. Images were acquired using a 20X 0.5 EC Plan-Neofluar objective on an epiflourescence microscope system (AxioVision Z1; Carl Zeiss, Thornwood, NY, USA) fitted with an Apotome slide module and a digital camera (AxioCam MRm).
4
Western Blot Analysis of Cellular Proteins
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Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40 Substitute). Protein concentration was determined using the Bradford reagent (Sigma Aldrich). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes by using Trans-Blot TurboTM Transfer Pack (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed using primary antibodies against Pgp, PON2, TXNIP, CYP D (Thermo Fisher Scientific), SOD2, OPA1 (BD Biosciences, St. Diego, CA, USA), TRPM7 (Bethyl, Montgomery, TX, USA), MagT1 (Abcam, Cambridge, UK), SIRT2 (Merck Millipore, Burlington, MA, USA), DRP1 (Cell Signalling, Danvers, MA, USA), β-actin and GAPDH (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) were used. The immunoreactive proteins were detected with ClarityTM Western ECL substrate (Bio-Rad) and images were captured with a ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with the software ImageJ (National Institute of Health, Bethesda, MD, USA). The Western blots shown are representative and the densitometric analysis was performed on three independent experiments ± SD.
5
Molecular Analysis of Dopaminergic Organoids
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Organoids were dissociated by a homogenizer and washed with 1× PBS. Homogenized organoids were extracted in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl in 50 mmol/L Tris [pH 8.0], Sigma-Aldrich; and 1× proteinase inhibitor mixture, Roche). The extracted protein was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with the following primary antibodies: NURR1 (1:200, Santa Cruz, sc-991), VMAT2 (1:1,000, Abcam, AB1598P), PITX3 (1:1,000, Invitrogen, 382850), pS129-α-synuclein (1:500, Abcam, AB9850), cleaved caspase-3 (1:500, Cell Signaling, 9661s), TXNIP (1:500, Thermo, 40-3700), Phospho-ERK1/2 (1:1,000, Cell Signaling, 4370s), ERK1/2 (1:1,000, Cell Signaling, 4695s), Phospho-p38 (1:500, Thermo, MA5-15177), p38 (1:500, Antibodies online, abin2957701), and β-actin (1:1,000, AbFrontier, LF-PA0207). Representative images are shown of western blots performed using Chemidoc TRS+ with Image Lab software (Bio-Rad).
6
Western Blot Analysis of HUVEC Lysates
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HUVEC were lysed in 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl2, 10 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5 mM EDTA and protein inhibitors, separated on SDS-PAGE and transferred to nitrocellulose sheets at 400 mA for 2h at 4 °C. Western blot analysis was performed using antibodies against P-HSP27, HSP27, DRP1, pDRP1Ser616, pDRP1Ser637, OPA1 (Cell Signaling Technology, Danvers, MA, USA), TXNIP, CYP D, ZO-1, VECAD (Thermo Fisher Scientific), HSP70 and Actin (Tebu Bio-Santa Cruz, Magenta, Italy), which were used as a control of loading. After extensive washing, secondary antibodies labeled with horseradish peroxidase (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) were used. The Super-Signal chemiluminescence kit (Thermo Fisher Scientific) was used to detect the immunoreactive proteins. All the experiments were performed at least three times, and a representative blot is shown. Densitometry of the bands from three blots was performed with the software ImageLab (Bio-Rad, Hercules, CA, USA), and the results are expressed as the mean ± SD.
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