Western Blot Analysis of NCYM, MYCN, and Other Proteins

Cells were lysed with RIPA buffer, Benzonase (Millipore, Billerica, MA, USA), and MgCl₂ at final concentrations of 25 U/μL and 2 mM, respectively, incubated at 37°C for 1 h, and centrifuged at 10,000 × g for 10 min at 4°C, after which the supernatant was collected. The supernatant was denatured in SDS sample buffer with or without 2-mercaptoethanol (reducing or non-reducing, respectively). Cell proteins were resolved by SDS-PAGE before being electroblotted onto polyvinylidene fluoride membranes. We incubated the membranes with the following primary antibodies for 60 min: anti-NCYM [1:1000 dilution (1 (link))], anti-MYCN antibody (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-Lamin B (1:1000 dilution; Millipore), anti-α-tubulin (1:1000 dilution; Cell Signaling Technology), anti-HA (1:1000 dilution; Cell Signaling Technology), and anti-actin (1:1000 dilution; Wako, Osaka, Japan). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG at 1:5000 dilution or anti-mouse IgG at 1:5000 dilution; both from Cell Signaling Technology), and the bound proteins were visualized using a chemiluminescence-based detection kit (ImmunoStar Zeta, Wako; ImmunoStar LD, Wako).