Indirect immunofluorescence assay.

Indirect immunofluorescence assay was carried out as described previously (68). Briefly, stable U2OS cell lines [KO+ZNF622(wt)-HA, KO+ZNF622(1-359), and KO+ZNF622(109-477)] were infected with HAdV-pVII-Flag (1 FFU/cell) or mock infected (infection medium without virus) for 48 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.1% Triton X-100 in PBS-T (PBS plus 0.01% Tween 20) for 15 min at room temperature. After blocking the cells with 3% BSA/PBS-T solution, the coverslips were subjected to immunofluorescence analysis using anti-Flag (Sigma, rabbit, 1:1,000) and anti-HA (BioLegend, mouse, 1:1,000) antibodies. Proteins were visualized using anti-mouse IgG(H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11029, 1:500) and anti-rabbit IgG(H+L) cross-adsorbed secondary antibody, and Alexa Fluor 594 (Thermo Fisher Scientific, A-11012, 1:500) secondary antibodies. Nuclei were counterstained with DAPI (1 μg/ml), and the coverslips were mounted using Fluoromount-G (Southern Biotech) mounting solution. Cells were visualized by a fluorescence microscope (Nikon Eclipse 90i), and the images were processed with NIS-elements (AR 3.10; Nikon) software. Immunofluorescence assays with U2OS cells expressing transfected Myc-NPM1 were performed as described above. Anti-Myc (Sigma, C3956, rabbit, 1:1,000) was used to detect the Myc-NPM1 protein.