Ecl enhanced chemiluminescence reagents

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)).

HEK293T cells expressing TMEM230 that were treated with either vehicle or inhibitors were harvested using lysis buffer (Cell Signaling). The resulting lysates were subjected to protein assays. An aliquot of 30 μg of protein from each sample was resolved on 4–12% NuPAGE Bis/Tris gels and transferred onto polyvinylidene difluoride membranes (Millipore). After being washed with TBST (TBS and 0.05% Tween-20) buffer, the membranes were blocked with 4% milk for 1 h and then incubated with various primary antibodies: anti-TMEM230 (1:500 dilution), anti-myc (1:1,000 dilution), rabbit anti-PARP (1:1,000 dilution), and rabbit anti-cleaved PARP (1:1,000 dilution), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-rabbit IgG (1:5,000 dilution), and goat anti-mouse IgG (1:5,000 dilution). Immunoblot signals were detected by ECL enhanced chemiluminescence reagents (PerkinElmer).