Zscan4

Cells were fixed in ice cold methanol/acetic acid (3:1). The fixed cells were dropped on microscope slides. Antigen retrieval was performed followed by blocking for 10 min at room temperature. Primary antibody was incubated overnight at 4 °C for the following antigens: ZSCAN4 (Origene, 1:1000), RNF20 (Cell Signaling, 1:2000), and Ubiquitin Lysine 48 (Millipore 1: 2000). The slides were washed and incubated with secondary antibodies conjugated with Alexa-488 or 568 at room temperature for 1 h and then treated with DAPI and To-Pro-3 to stain the nuclei. Slides were mounted and visualized by a Zeiss 510-confocal microscope. Co-localization analyses were performed by ImageJ software [16 (link)].

Nuclear proteins were fractionated using Nuclear Extraction Kit following manufacture's protocol (Active Motif). Total cell lysate was prepared in RIPA buffer and sonicated. For the detection of endogenous ZSCAN4 in Tu167 cells, cells were harvested by accutase (Millipore) and Cytoskeleton buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 1 mM EGTA and 0.5% Triton X100) was used to fractionate cytosolic proteins. Then, pellets were lysed in urea solution (8 M Urea in 0.01 Tris pH 8 + 0.1 M NaH₂PO₄) and sonicated. Nuclear proteins were electrophoresed in 8% polyacrylamide gels and transferred to a PVDF membrane. Immunoblot was performed using the following primary antibodies: ZSCAN4 (Origene; 1:1000), GAPDH (Santa Cruz; 1:5000), Actin (Sigma; 1:500), Lamin B (Santa Cruz; 1:2000) and with HRP (horseradish peroxidase) conjugated secondary antibodies (Millipore; 1:5000). Protein bands were detected using Pierce ECL Western Blotting Substrate (Thermo Scientific). SuperSignal West Femto (Thermo Scientific) was used to detect endogenous ZSCAN4 in Tu167 cells. All immunoblots shown represent at least 3 independent experiments.