Parp 1 h 250

Cells were harvested in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Protein lysate (20 μg) was resolved in SDS-polyacrylamide gels, transferred to PVDF membrane and then probed with antibodies specific to: folate receptor alpha (FR-α) (ab125030; Abcam, Cambridge, MA, USA); Cyclin D1 (72-13G), CDK4 (C-22), and PARP-1 (H-250) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); p21^WAF−1 (12D1) and Bcl-2 (2876S) (both from Cell Signaling Technologies, Danvers, MA, USA); and Caspase-3 (610322; BD Transduction Laboratories, San Diego, CA, USA). The membranes were next incubated with the respective secondary antibodies (Thermo Fisher Scientific). Anti-β-actin (AC-15; Abcam) and anti-β-tubulin (T5293; Sigma-Aldrich, St. Louis, MO, USA) antibodies were used as internal loading controls. The protein bands were quantitated using ImageJ software.