After incubation of 5×106 HCT116 cells with or without 500 μM Olsa-RVRR, olsalazine, CTR1, or CTR2 at 37 °C for 3 h, cells were washed three times and then cultured in fresh McCoy’s 5A (modified) medium. Forty-eight hours later, the nuclear proteins for each group were extracted using a nuclear extraction kit (Epigentek). After measuring the protein concentration using a Bradford protein assay, a DNA methyltransferase activity/inhibition assay kit (Epigentek) was used to detect DNMT activity. The protein content in each well was 8 μg (except for blank ones).
Dna methyltransferase activity inhibition assay kit
Manufactured by Epigentek
Sourced in United States
The DNA methyltransferase activity/inhibition assay kit is a laboratory tool used to measure the activity or inhibition of DNA methyltransferase enzymes. It provides a quantitative method for assessing the functional status of these enzymes.
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4 protocols using dna methyltransferase activity inhibition assay kit
1
Olsalazine Modulates DNMT Activity
2
Quantifying DNA Methyltransferase Activity
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The EpiQuik™ Nuclear Extraction Kit (Epigentek Group, USA) was used to prepare the nuclear extracts. DNMT1 activity was measured by a non-radioactive protocol using the DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek) as instructed by the manufacturer, based on the following formula: DNMT activity (OD/h/mg) = (No inhibitor OD-Blank OD) 1000/Protein amount (µg) × hour. The relative DNMT1 activity was normalized to the activity of the control group.
3
Olsalazine Modulates DNMT Activity
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After incubation of 5×106 HCT116 cells with or without 500 μM Olsa-RVRR, olsalazine, CTR1, or CTR2 at 37 °C for 3 h, cells were washed three times and then cultured in fresh McCoy’s 5A (modified) medium. Forty-eight hours later, the nuclear proteins for each group were extracted using a nuclear extraction kit (Epigentek). After measuring the protein concentration using a Bradford protein assay, a DNA methyltransferase activity/inhibition assay kit (Epigentek) was used to detect DNMT activity. The protein content in each well was 8 μg (except for blank ones).
4
Nonradioactive DNMT1 Activity Assay
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DNMT1 activity in all nuclear extract samples was measured nonradioactively by using DNA Methyltransferase Activity/Inhibition Assay Kit as instructed by the manufacturer (Epigentek, Group, USA). Relative DNMT1 activity was normalized to that of MHCC97H (or SK-Hep-1 and Hep G2) cells or LCSCs (or LCSLCs).
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