51 mm polycarbonate centrifuge tubes

Assays were performed as described (Nitiss et al., 2012 (link)). Cells were lysed with 1% sarkosyl, and the DNA was sheared using a 1 ml syringe and 25G/8 gauge needle. Lysates were passed 15 times through the needle. Lysates (1.5 ml) were then layered atop a 2 ml CsCl solution in 13 × 51 mm polycarbonate centrifuge tubes (Beckman) and spun at 71,000 rpm in an SLA-100.3 rotor (Beckman) for 16–20 h at room temperature. Pellets were washed with 70% ethanol and resuspended in TE buffer (pH 7.5). Indicated amounts of DNA were applied to a nitrocellulose membrane using a slot-blot apparatus (Bio-Rad). The membranes were then assessed for the presence of Top2Bccs using standard western blotting procedures. The gel analyzer routine of ImageJ software was used to subtract background and quantify the optical density of the bands. The optical densities of the three DNA amounts were averaged. Each dot in the bar graph (individual data and mean ± s.e.m.) represents the result of an independent experiment/biological replicate performed on different days using neurons cultured from independent mouse litters. Number of dots per graph represent the n for every experiment, and p-values < 0.05 for relevant comparisons are indicated in the graphs. The densitometric data were analyzed by one-way ANOVA with Tukey’s post-hoc test.

ICE assays were performed essentially as described (Nitiss et al., 2012 ). Cultured primary neurons were lysed with 1% sarkosyl, and the DNA was sheared using a 1 ml syringe with a 25G/8 gauge needle. Lysates were passed 10 times through the needle. Lysates (2 ml) were then layered atop a 2 ml CsCl solution in 13 × 51 mm polycarbonate centrifuge tubes (Beckman) and spun at 71,000 rpm in an SLA-100.3 rotor (Beckman) for 12 hr at room temperature. Pellets were washed with 70% ethanol and resuspended in TE buffer (pH 7.5). Indicated amounts of DNA were applied to a nitrocellulose membrane using a slot-blot apparatus (Bio-Rad) following manufacturer’s instructions. The membranes were then analyzed for Topo IIβ using standard western blotting procedures.