Normal goat serum (ngs)

Cells were grown on poly-L-lysine (Wako, Osaka, Japan)-coated coverslips and fixed with 2% paraformaldehyde (Wako) in PBS for 10 min. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min and blocked with 5% normal goat serum (NGS; Chemicon, Temecula, CA) in PBS for 1 h. Cells were stained with primary antibodies (1% NGS in PBS) for 2 h and secondary antibodies (1% NGS in PBS) for 2 h, and then counterstained with DAPI (Roche) and mounted in PPDI [80% glycerol in PBS, 1 mg/ml paraphenylenediamine (11873580001, Roche)]. Images were recorded with a DeltaVision microscope using 60×1.40 and 100×1.35 Plan Apo objective lenses. For lamin-B staining, 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS was used as blocking buffer, and 1% BSA in PBS was used for dilution of antibodies (Fig. S1B).

For immunocytochemistry, primary neurons were fixed in pre-chilled methanol at −20 °C for 10 min, washed in PBS (3 × 5 min), permeabilized in 0.2% Triton X-100 (St. Louis, MO, USA) in 1× PBS (20 min at RT), and blocked for 1 h in 10% Normal Goat Serum (Roche Diagnostics) in 1× PBS. Primary antibodies were incubated overnight at 4 °C in blocking solution. The primary antibodies used were CERKL(ex2) and CERKL(ex5), and were obtained in-house against epitopes encoded in exon 2 or exon 5 of the mouse Cerkl gene respectively [16 (link)]. After incubation, coverslips with cells were rinsed in 1× PBS (3 × 5 min), incubated with the corresponding secondary antibodies conjugated to Alexa Fluor 488 (Life Technologies, Grand Island, NY, USA) (1:500) at RT (1 h) in blocking solution. Nuclei were stained with DAPI (Roche Diagnostics, Indianapolis, IN, USA) (1:1000), washed again in 1× PBS (3 × 5 min), and mounted in Mowiol 4–88 (Merck, Darmstadt, Germany).

In immunocytochemistry experiments, cells were fixed in pre-chilled methanol at −20 °C for 10 min, washed in PBS 1× (3 × 5 min), permeabilized in 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (20 min at RT), and blocked for 1 h in 10% Normal Goat Serum (Roche Diagnostics, Indianapolis, IN, USA) in PBS. Primary antibodies were incubated overnight at 4 °C in a blocking solution. The primary antibodies used were: p65 (Cell Signaling Technology, Danvers, MA, USA; 8242, 1:500) and TUBULIN (Sigma-Aldrich, Saint Louis, MO, USA; T5168, 1:500). After incubation, coverslips with cells were rinsed in PBS 1× (3 × 5 min), incubated with the corresponding secondary antibodies (AlexaFluor 488 anti-Mouse) (Thermo Fisher Scientific, Rockford, IL, USA; A11017; 1:500), AlexaFluor 488 anti-Rabbit (Thermo Fisher Scientific, Rockford, IL, USA; A11070; 1:500) at RT (1 h) in blocking solution. Nuclei were stained with DAPI (Roche Diagnostics, Indianapolis, IN, USA) (1:1000), washed again in PBS 1× (3 × 5 min), and mounted in Mowiol 4–88 (Merck, Darmstadt, Germany).