Western Blot Analysis of Protein Expression

Cells were collected using cell lysis buffer (Cell Signaling, Inc, CST-9803S) with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin, and 1 mM PMSF. Cell lysates from each group were subjected to protein assays. An aliquot of 30 μg protein from each sample was resolved on 4–12% NuPAGE Bis/Tris gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 4% milk for 1 h, then incubated with various primary antibodies as indicated in Reagents section: anti-Flag (1:1,000 dilution), rabbit anti-PARP (1:1,000 dilution), and rabbit anti-cleaved PARP (1:1,000 dilution) followed by HRP-conjugated secondary antibodies: goat anti-Rabbit IgG (1:5,000 dilution) or goat anti-Mouse IgG (1:5,000 dilution). Protein bands were detected by ECL enhanced chemiluminescence reagents (PerkinElmer) as described previously (Li et al., 2014 (link); Yang et al., 2018 (link); Arbez et al., 2020 (link)).