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COS-7 cells were transfected with a pCAG-Dpy19L1 plasmid. Forty-eight h later, they were lyzed with Cell-LyEX MP (TOYO INK). Cell lysates were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore) by wet transfer method (Bio-Rad). Following blocking with 5% nonfat dry milk, the membrane was incubated with rabbit anti-Dpy19L1 (N-ter, 1:500, Abcam), rabbit anti-Dpy19L1 (C-ter, 1:500, Abgent), or mouse anti-α-Tubulin (1:4000, Sigma) overnight at 4°C and then reacted with the secondary antibody conjugated to horseradish peroxidase (1: 10000, MBL). Signals were developed using Immunostar LD (Wako) and scanned with a C-DiGit Blot Scanner (LI-COR).

Cultured cells were fixed with 4% paraformaldehyde (PFA) in 0.01M PBS at room temperature or methanol/acetone (1:1) at −30°C for 15 min, and then rinsed in PBS. For F-actin staining, transfected cells were fixed with 4% PFA, followed by permeabilization with 0.5% TritonX-100 in PBS for 10 min. After blocking with 10% normal goat serum and 0.1% TritonX-100 in PBS, cells were reacted with primary antibodies overnight at 4°C. After washing, cells were labeled with species-specific secondary antibodies conjugated to Alexa Fluor 488 or 594 (1:1000, Life Technologies) and counterstained with Hoechst 33342 (Sigma) or DRAQ5 (Abcam). Primary antibodies used were: rat anti-GFP (1:2000, Nacalai Tesque), rabbit anti-Dpy19L1 (C-ter, 1:100, Abgent; N-ter, 1:250, Abcam), chicken anti-Calreticulin (1:1000, Abcam), mouse anti-Calreticulin (1:200, Abcam), rabbit anti-active Caspase-3 (1:1000, BD Biosciences), Phalloidin (CF dye conjugates, 1:200, Biotium) and mouse anti-α-Tubulin (1:2000, Sigma). Pictures were taken with a digital camera (DP72, Olympus). Confocal images were captured with a confocal laser scanning microscope (FV1200). Intensity profile analysis was performed using FV10-ASW software (Olympus). For colocalization analysis, the scatter plots of red and green pixel intensities of confocal images were created using Image J/Fiji software.