Western blot analysis of mouse tissues and cells

Mouse testis were snap-frozen in liquid nitrogen and subsequently lysed in RIPA buffer (150 mM NaCl, 100 mM Tri pH 7.5, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycolate) containing protease and phosphatase inhibitors (ROCHE) using the precellys 24 tissue disruptor (Berlin technologies). Similarly, primary fibroblasts from Pole4^+/+ and Pole4^-/- embryos (MEFs) in a Trp53^+/+, ^+/- or ^-/- background were lysed in RIPA containing protease and phosphatase inhibitors. Lysates were clarified by centrifugation (12.300 rpm 30 min at 4°C) and protein concentration was estimated by BRADFORD assay (SIGMA). Equal amounts of proteins were loaded on NuPAGE 4-12% Bis-Tris gels and transferred onto nitrocellulose membrane (Amersham). Membranes were blocked in 5% milk in PBST (PBS-Tween 0.1%) and incubated with primary antibodies and HRP-conjugated secondary antibodies.