Samples were suspended in lysis buffer (250 mM Sucrose, 50 mM Tris pH 7.4, 1 MgCl, and 0.2 % (v/v) Triton X-100). The cell lysates were then centrifuged at 5,000 g at 4°C for 15 min. A total of 20 μg of protein was analyzed using standard immunoblotting analysis. Membranes were blocked overnight in 5% milk in blocking buffer (0.2% Triton X-100 in PBS) at 4°C. The primary antibodies used were; Glutamine synthetase (GS, Abcam) 1:1,000, CYP3A4 (Abcam) 1:1,000, carbamoyl-phosphate synthase 1 (CPS1, Abcam) 1:1,000, Arginase 1 (Arg1, Abcam) 1:1,000, β-actin (Abcam) 1:1,000.
Cyp3a4
Manufactured by Abcam
Sourced in United Kingdom
CYP3A4 is a member of the cytochrome P450 enzyme family, which play a crucial role in the metabolism of various drugs and other xenobiotics. CYP3A4 is the most abundant and versatile of the human cytochrome P450 enzymes, responsible for the oxidative metabolism of a wide range of substrates.
Automatically generated - may contain errors
Lab products found in correlation
Cyp2e1, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Aml12, by American Type Culture Collection (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Percoll, by Merck Group (1 mentions)
Brl 3a, by American Type Culture Collection (1 mentions)
Reverse transcript enzyme, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
Goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
8 protocols using cyp3a4
1
Liver Protein Expression Analysis
2
CYP3A4 Protein Expression Quantification
3
Hepatoprotective Effects of 2-Hydroxybutyrate
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Male SD and Wistar rats (160–180 g) and C57 mice (18–22 g) were purchased from Shanghai SLAC Laboratory Animal Company and housed at constant temperature (24 ± 2 °C) with a 12 h light-dark cycle. BRL3A (ATCC number: CRL-1442) and AML12 (ATCC number: CRL-2254) cells were purchased from ATCC (Manassas, MA, USA). Vac was purchased from Lilly Company (USA). AP, 2-HB, phenacetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium (±)-2-hydroxybutyrate-2,3,3-d3 (d3-2-HB) was purchased from C/D/N Isotopes Inc (Canada). ALT, AST kits were purchased from Nanjing Jiancheng Bioengineering Institute (China). GSH & GSSG and CCK-8 kits were purchased from Beyotime Biotechnology (China). Gut bacterial DNA extraction kit was purchased from QIAGEN Company (Germany). TRIzol reagent, reverse transcript enzyme and SYBR Green master mix were purchased from Invitrogen (Carlsbad, CA). CYP2E1 (Cat: ab28146, Lot:GR324351-11), CYP3A4 (Cat: ab3527, Lot: GR3210692-3), NQO-1 (Cat: ab2346, Lot: GR3550-44) primary antibodies were purchased from Abcam (USA). Collagenase type I (Cat: LS004196) was purchased from Washington Biochemical Corporation. Percoll (Cat: P1644) was purchased from Sigma-Aldrich.
4
Quantification of Liver Protein Biomarkers
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Liver protein was extracted with a commercial lysis buffer (Shanghai beyotime Biotechnology Company) added with 1% of phosphatase inhibitor cocktail 2 and phosphatase inhibitor cocktail 3 (Sigma) according to well-established protocols. The concentration of total protein was determined with Pierce™ BCA protein assay kit (Thermo Scientific). Then the samples were adjusted to the same concentration and mixed with loading buffer, and heated at 100 °C for 10 min. A total of 40 μg protein was loaded into each lane and separated by 10% SDS-PAGE gel, and then transferred to a PVDF membrane (Millipore). The membranes were blocked with 10% milk at room temperature for 90 min and then washed with TBST (20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween20, pH7.5) for three times at 10 min interval, following the incubation with primary antibodies for NQO-1 (Abcam), CYP2E1 (Abcam) and CYP3A4 (Abcam) overnight at 4 °C. The membranes were washed with TBST for three times, and incubated with the HRP-conjugated secondary antibodies for 2 h at room temperature. The membranes were exposed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and the bands were quantified with Amersham Imager 600 system (General Electric Company, USA).
5
Characterization of ADSC Protein Expression
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ADSCs were cultured for 21 days with different described condition and fixed with 4% of paraformaldehyde (Sigma Aldrich Chemie GmbH, Hamburg, Germany) for 30 min at room temperature. After permeabilization by 0.1% Triton X-100 (Thermo Fisher Scientific, Grand Island, NY, USA) -PBS, cells were washed in PBS three times for 5 min. After washing, ADSCs were incubated with 3% bovine serum albumin (BSA)—0.1% Triton X-100 in PBS (Thermo Fisher Scientific, Grand Island, NY, USA) for 30 min and then exposed overnight at 4 °C to the primary anti-mouse monoclonal antibodies directed against activating signal cointegrator-1 (ASC-1) (Santa Cruz Biotechnology, Heidelberg, Germany), proton-coupled amino acid transporter (PAT2) (Santa Cruz Biotechnology), CYP27A1 (Abcam, Cambridge, UK), and CYP3A4 (Abcam, Cambridge, UK). Finally, cells were washed two times in PBS for 5 min and stained at 37 °C for 1 h in the dark with the fluorescence-conjugated goat anti rabbit IgG secondary antibody (Life Technologies, USA) and goat anti mouse IgG secondary antibody (Life Technologies, USA). Nuclei were labelled with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Grand Island, NY, USA). All microscopy analyses were performed with a confocal microscope (TCS SP5, Leica, Nussloch, Germany).
6
Hepatic Protein Expression Analysis
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Total proteins were extracted from frozen hepatocytes and hepatic samples, and then the protein concentrations were measured by BCA protein assay (Solarbio, China). The samples were separated on SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.2% Tween-20 at ambient temperature for 1 h, followed by incubation with following antibodies: GAPDH (1:5000; Proteintech, China), ALB (1:2000; Proteintech, China), CYP3A4 (1:2000; Abcam, UK), alpha 1 antitrypsin (AAT, 1:2000; Abcam, UK), cytokeratin 18 (CK-18, 1:2000; Abcam, UK), Bax (1:2,000; Proteintech, China), and Bcl-2 (1:2000; Proteintech, China). The membrane was treated with HRP-conjugated goat anti-rabbit secondary antibody (1:3000; Cell Signaling Technology, USA) for 1 h at room temperature. The protein bands were visualized with an ECL chemiluminescence solution (Millipore, USA) and imaged using the Imaging System (BIO-RAD, USA). Protein expression levels were determined by densitometry using Image J software.
7
Characterization of CYP Enzymes
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After transfections, cells were lysed with ice-cold RIPA lysis buffer (ThermoFisher Scientific, Carlsbad, CA) containing protease inhibitors (Pierce Protease Inhibitor Mini Tablets, Rockford, IL and Mammalian Protease Arrest, GBiosciences, St. Louis, MO) and centrifuged at 12,500 rpm for 10 min at 4 °C. Cell supernatants were used for immunoblots. Proteins were quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Carlsbad, CA) and 20 ug of protein was loaded on to precast polyacrylamide Bis-tris 10% gels (GenScript, Piscataway, NJ). The membranes were blocked with 5% skim milk and incubated with primary antibodies against CYP2E1, CYP1A2 and CYP3A4 (Abcam, Cambridge, MA) and β-actin (Sigma, St. Louis, MO). Next membranes were incubated with HRP-conjugated secondary antibodies to goat anti-rabbit IgG H&L (Abcam, Cambridge, MA). The protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Carlsbad, CA) and densitometry was performed with the ImageJ software program (NIH, Bethesda, MD).
8
Assessing Cytoprotective Mechanisms in APAP-Induced Toxicity
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4 × 105 cells were plated on each 6 cm dish for 48 h prior to APAP treatment at 32°C for 24 h. Next, after hypothermia or following rewarming for 24 h at 37°C, cells were lysed and the supernatants were collected to measure the activity of CYP3A4 (Abcam, Cambridge, UK) and glutathione-S-transferase (GST) (Abcam, Cambridge, UK) according to manufacturer’s instructions. For CYP3A4 activity, the fluorescence intensity was measured at an excitation wavelength of 535 nm and at an emission wavelength of 587 nm in a kinetic mode for 45 mins while for GST activity, the absorbance was measured at 340 nm in a kinetic mode for 20 mins on a microplate reader. Fold changes in fluorescence intensity, representative of CYP3A4 activity, were determined by normalizing against the negative control i.e. APAP-free treatment with continuous 37°C incubation. A total of 3 biological replicates was performed.
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