Western-blot analysis was carried out as described (Kausar et al. 2012 (link)). Briefly, microsomal proteins were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. After blocking with 5% non-fat dry milk in blocking solution, the membrane was incubated with CYP1A1 (1:4,000 dilution), CYP1A2 (1:3,000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) and CYP1B1 (1:1,500 dilution) (Abcam, Cambridge, MA) antibody overnight at 4°C. The membrane was then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA), and the immuno-reactive bands were visualized using the Pierce chemiluminescent substrate kit (Thermo Scientific, Rockford, IL). To ensure equal protein loading, each membrane was stripped and re-probed with β-actin antibody (Sigma-Aldrich, St Louis, MO) to normalize for differences in protein loading.
Cyp1a2
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom, United States
CYP1A2 is a cytochrome P450 enzyme that plays a role in the metabolism of various compounds, including drugs and environmental toxins. It is involved in the oxidative metabolism of a wide range of substrates.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Cyp1a1, by Santa Cruz Biotechnology (2 mentions)
Cyp1b1, by Abcam (2 mentions)
Pierce chemiluminescent substrate kit, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Gpr125, by Abcam (1 mentions)
Oct3 4, by Santa Cruz Biotechnology (1 mentions)
Sox17, by R&D Systems (1 mentions)
Hnf4α, by Santa Cruz Biotechnology (1 mentions)
Nanog, by Cosmo Bio (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Mscgm mesenchymal stem cell growth medium bulletkit, by Lonza (1 mentions)
Anti human cd45 fitc, by BD (1 mentions)
Anti human cd34 fitc, by BD (1 mentions)
5 protocols using cyp1a2
1
Western Blot Analysis of CYP Enzymes
2
Protein Expression Analysis in Grafts
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The grafts were minced thoroughly and lysed with RIPA buffer (Santa Cruz) on ice. Western blots were performed according to the protocol as described previously [33 (link)]. Briefly, the concentration of total proteins was measured first by BCA kit (Dingguo Company, China). Twenty micrograms of cell lysates were resolved by SDS-PAGE (Bio-Rad Laboratories, Richmond, CA). The chosen antibody included CYP1A2 (Santa Cruz), AAT (Bethyl), AFP (alpha-fetoprotein) (Santa Cruz), ALB (Santa Cruz), PCNA (Santa Cruz), GPR125 (Abcam), and ACTB (Proteintech). The dilutions, resources, and companies of primary and secondary antibodies for Western blots were shown in Table 2 . After extensive washes in PBS, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA).
3
Western Blot Analysis of CYP Enzymes
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Western-blot analysis was carried out as described [17] (link). Briefly, microsomal proteins were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. After blocking with 5% non-fat dry milk in blocking solution, the membrane was incubated with CYP1A1 (1:4000 dilution), CYP1A2 (1:3000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) and CYP1B1 (1:1500 dilution) (Abcam, Cambridge, MA) antibody overnight at 4 °C. The membrane was then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA), and the immuno-reactive bands were visualized using the Pierce chemiluminescent substrate kit (Thermo Scientific, Rockford, IL). To ensure equal protein loading, each membrane was stripped and re-probed with β-actin antibody (Sigma–Aldrich, St Louis, MO) to normalize for differences in protein loading.
4
iPSC Generation and Characterization
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The human STEMCCA/TAT-Cre bundle used for iPSC generation was purchased from Sigma-Aldrich (SCR545-CRE, Louis, MO, USA). Antibodies against KLF4 (Santa Cruz Biotechnology, sc-20691, Dallas, TX, USA), OCT3/4 (Santa Cruz Biotechnology, sc-5279), SOX2 (Cell Signaling Technology, 4900, Danvers, MA, USA), c-MYC (Santa Cruz Biotechnology, sc-764), NANOG (Cosmo Bio, REC-RCAB0004PF, Tokyo, Janpan), SOX17 (R&D Systems, AF1924), albumin (Thermo Fisher Scientific, A80-229A, Waltham, MA, USA), AFP (Abcam, ab3980, Cambridge, UK), HNF4α (Santa Cruz Biotechnology, sc-8987), ASGR1 (Santa Cruz Biotechnology, sc-52623), CYP1A2 (Santa Cruz Biotechnology, sc-53241), and β-actin (Santa Cruz Biotechnology, sc-47778) were used.
5
Stem Cell Differentiation Assay
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Recombinant human LIGHT (rhLIGHT) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-human CD90-fluorescein isothiocyanate (CD90-FITC), anti-human CD44-FITC, anti-human CD105-FITC, anti-human CD34-FITC, anti-human CD45-FITC, anti-human CD19-FITC, anti-LTβR-PE, and FITC Mouse immunoglobulin G (IgG)-isotype control antibodies were purchased from BD Biosciences (San Diego, CA, USA). The Mesenchymal Stem Cell Growth Medium BulletKit™ (MSCGM™) was obtained from Lonza (Basel, Switzerland). For western blotting, antibodies against albumin, SRY-box transcription factor 17 (SOX17), forkhead box A2 (FOXA2), cyclin dependent kinase 1 (CDK1), CDK6, cyclin B1, cyclin D3, B-cell lymphoma-extra large (Bcl-xl), inhibitor of κB-α (IκB-α), protein kinase B (AKT), phospho-AKT, signal transducer and activator of transcription 3 (STAT3), phospho-STAT3, STAT5, phospho-STAT5, Rabbit IgG-horseradish peroxidase (IgG-HRP), and Mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cytokeratin-18 (CK-18), CK-19, cytochrome P450 family 1 subfamily A member 1 (CYP1A1), CYP1A2, CYP3A4, CDK2, CDK4, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). All reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).
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