Western Blot Protein Analysis

Western blotting analysis was performed using standard protocols as published elsewhere [10 (link), 14 (link)]. Briefly, protein lysates were extracted from the cells (1 × 10⁷ cells) using a Qproteome Mammalian Protein Prep Kit (Qiagen), and the lysates were applied to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for separation. Proteins were then transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were probed with primary and secondary antibodies using standard techniques. Anti-FPGS (cat. no. ab184564; Abcam, Cambridge, UK), anti-DHFR (cat. no. 872442; R&D Systems, Minneapolis, MN, USA), anti-caspase 3 (cat. no. 9665; Cell Signaling Technology, Massachusetts, USA), anti-PARP (cat. no. 9542; Cell Signaling Technology), anti-cleaved PARP (cat. no. 9541; Cell Signaling Technology), and anti-β-actin (cat. no. A2066l Sigma-Aldrich Japan) antibodies were used as primary antibodies, and anti-rabbit polyclonal antibodies (cat. no. 7074; Cell Signaling Technology, Tokyo, Japan) were used as secondary antibodies. Protein detection and quantification were performed using Amersham ECL Prime Western Blotting Detection Reagent and an ImageQuant LAS4000mini system (GE Healthcare Life Sciences, Little Chalfont, UK).