Nytran n nylon blotting membrane

To detect the amount of DNA adducts in the control and TFEB silenced IGROV-CP20 cells, DNA was isolated using the Quick-DNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA) and 1 ug DNA was spotted on a piece of Nytran N nylon blotting membrane (GE Healthcare Life Sciences, Boston, MA, USA). Dot blot experiments were performed using an ant adduct antibody (ICR4, Merck, Darmstadt, Germany) following the previously-described procedure [12 (link)].

To detect the amount of DNA adducts in control and treated cells, the DNA was extracted from the cells using Quick-DNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA). Equal amounts of DNA from each specimen were spotted on a piece of Nytran N nylon blotting membrane (GE Healthcare Life Sciences, Marlborough, MA, USA). The membrane was incubated with a primary antibody that recognized Pt-induced DNA adducts (ICR4, Merck, Millipore), then washed with TBST and incubated with an HRP-conjugated secondary antibody according to the manufacturer’s instructions. Chemiluminescent signals were captured using Chemidoc Amersham Imager 600 and quantified using ImageJ software.