Orf45

Cells were seeded on coverslips in a 24-well plate, washed with PBS (Biological Industries, Israel), and fixed with 4% formaldehyde in PBS for 25 min at room temperature. After fixation, cells were washed with PBS, and permeabilized and blocked in PBS containing 0.2% Triton X-100% and 1% BSA. Slides were incubated with primary antibodies to UBF (Sigma/Santa Cruz, CA, USA), Fibrillarin (Abcam, Cambridge, UK), NPM (Abcam, Cambridge, UK), Nucleolin (Abcam, Cambridge, UK), RPA194 (Santa Cruz, CA, USA), PICT-1 (Santa Cruz, CA, USA), PML (Santa Cruz, CA, USA), ORF45 (Santa Cruz, CA, USA), ORF65 [64 (link)] (a kind gift from Shou Jiang Gao) or ORF59 (a kind gift from Prof. Bala Chandran, University of South Florida) [65 (link)] at 40 C, followed by incubation with secondary conjugated antibodies (Rhodamine, Cy3, Alexa Fluor 647 or Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at room temperature. To stain the nuclei, cells were incubated for 10-min with 0.05 µg/mL Hoechst dye (Sigma) in PBS. The slides were mounted with anti-fading medium (1% n-Propyl gallate, 90% Glycerol in PBS). Cells were examined and photographed under a confocal laser-scanning microscope (Leica SP8 Confocal Live Microscope).

The whole-cell lysates were resolved by SDS–PAGE, transferred to a polyvinylidene fluoride membrane and probed with primary antibodies against FLAG-M2 (1:2000; Sigma), MYC (1:2000; made in our laboratory), ORF45 (1:500; made in our laboratory), GFP (1:500; Santa Cruz Biotechnology), PARP-1 (1:1000, BD Biosciences) or α-tubulin (1:2000; Sigma). A goat anti-rabbit or goat anti-mouse immunoglobulin G antibody conjugated with horseradish peroxide (a secondary antibody; Santa Cruz Biotechnology) was detected using ECL plus Western blotting detection reagents (ELPIS), and the signals were analyzed on LAS-4000, a chemiluminescent image analyzer (Fujifilm).