Figure 5

Analysis of CCDC6 expression in normal human testis and testicular germ cells tumors. (A) 40 ug of total tissue lysates were resolved on 10% SDS-PAGE transferred onto nitrocellulose filters and western blotted with α-CCDC6 antibody. NT, normal testis; lanes 2–6: seminomas; lane 7, teratoma; lane 8, embryonal carcinoma; lane 9, TCAM2 seminoma cell line. Anti-actin hybridization is shown as loading control. The blots are representative of three independent experiments. (B) Top: CCDC6 relative expression in TCAM2 and GC1 cells analysed by qRT-PCR. Data are the mean +/− SD of three independent experiments. Bottom: CCDC6 expression in the unique TCAM2 seminoma cells and GC1 Cells. Whole lysates were detected by western blotting with anti-CCDC6 antibody. (C) the activation of the molecules of the DNA Damage Response were monitored using the anti-pS1981_ATM (pATM), the anti-pT68_Chk2 (pChk2), and the anti-pS139_H2AX (γH2AX). Total ATM, Chk2 and H2AX are shown. Before lysis, the cells have been treated 1 hour with H₂O₂ (10 μM), and after washing out the hydrogen peroxide the cells have been left an additional hour before the lysis. Anti-tubulin hybridization is shown as loading control.