Anti cleaved parp

MDA-MB-231 cells were plated in a 6-well plate at a concentration of 0.7 × 10⁵ cells/mL and incubated overnight. The cells in each well were treated differently: either with DMEM media only (control), with 15 µM cisplatin, with 4% v/v UD alone, or co-treated with 15 µM cisplatin and 4% v/v UD. After 48 h, cells were lysed using the RIPA buffer and protease inhibitor cocktail (MP Biochemical, California, USA). Proteins were collected, and quantification was performed using the Lowry method. Proteins were resolved by SDS-PAGE and the gels were blotted onto PVDF membranes. The membranes were blocked with 5% skimmed milk and then incubated with primary antibodies: anti-actin (Santa Cruz, Dallas, TX, USA), anti-Bax (Invitrogen, Waltham, MA, USA), anti-Bcl2 (Invitrogen, Waltham, MA, USA) and anti-cleaved Parp (Invitrogen, Waltham, MA, USA). After washing, the membranes were treated with the appropriate secondary antibodies for 1 h at room temperature (BioRad, Hercules, CA, USA). The membranes were washed again, and images were generated using the Clarity™ Western ECL Substrate on the ChemiDoc machine (BioRad, Hercules, CA, USA). To quantify blot bands and to calculate the relative expression of proteins, the ImageJ software was used [57 (link)].