Ab735

Male germ cells from wild-type mice and P63^(+/−) mice were lysed with RIPA buffer (BiotechWell, Shanghai, China) for 30 min on ice. Cell lysates were cleared by centrifugation at 12,000×g for 15 min at 4 °C, and the protein concentrations were measured by BCA kit (DingGuo ChangShengBiotech, Beijing, China). In total, 30 mg of cell lysate from each sample were separated using 10% SDS-PAGE (Bio-Rad Laboratories) and transferred to nitrocellulose membranes for 2 h at room temperature. The membranes were blocked using 5% nonfat dry milk in TBS-T for 1 h at room temperature. After washing with TBS-T, the membranes were incubated with antibodies against P63 (Abcam, ab735, 1:500) and ACTB (Protein tech, catalog no: HRP-60008, dilution: 1:5000) overnight at 4 °C. After extensive washes, the membranes were incubated with horseradish peroxidase-conjugated immunoglobulin G (IgG) (Santa Cruz Biotechnology) at a 1:2000 dilution for 1 h at room temperature. The membranes were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA, USA), and densitometric analyzes were processed with Adobe Photoshop 8.0. The relative level of P63 protein was normalized to the expression of ACTB.

MEECs were grown to an appropriate density and fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X-100 for 15 min, and blocked with 5% bovine serum albumin for one hour at room temperature. After blocking, cells were labeled with the primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, rabbit anti-CD44, Proteintech, 15675-1-AP; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321), and the secondary antibodies (1:100, fluorescently labeled goat anti-mouse lgG-cy3, BA1031, Boster company, Wuhan, China) according to the manufacture’s protocol. DAPI (0.5 mg/ml, D3571, Thermo Fisher) was used to stain the nucleus. Then, the fluorescence was detected by Leica DM4000B fluorescence microscope.