Nup98

Cells were harvested from wild-type and ATM-null BM culture at indicated days, and then lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA (pH 8.0), 20 mM NaF, 1 mM Na₃VO₄, 1% NP40, 0.5 mM dithiothreitol) in the presence of protease–inhibitor mix (leupeptin, aprotinin, and PMSF, 10 μg/ml, respectively). Total cell lysate (10 μg) was loaded and separated by 6∼15% SDS-PAGE gels. Antibodies for ATM (GeneTex, Irvine, CA, USA), Akt, Erk, H2AX (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p38, STAT5, NUP98, Histone H3, eIF4E, and 4EBP1 (Cell Signaling Technology, Danvers, MA, USA) were used to detect total proteins. Specific anti-phosphorylation antibodies were used against phosphor-H2AX (γH2AX, Novus Biologicals, Littleton, CO, USA), phospho-Akt (Ser473), phospho-p44/p42 (Thr202/Tyr204), phospho-p38 (Thr180/Try182), and phosphor-STAT5 (Tyr694, all from Cell signaling Technology). The antibody for phosphorylated mouse ATM (S1987) was purchased from R&D systems (Minneapolis, MN, USA). Specificities of ATM antibodies used in this study are shown in Supplementary Figure S3. Anti-actin antibody (Santa Cruz Biotechnology) was used to validate protein amount. To analyze the localization of ATM in BM, cytoplasmic extract and nuclear extract were prepared as described.^{50 (link)}