DNA replication stress induces soluble H2AX. (A) Immunoblot analysis for GFP of U-2 OS cells stably transfected with empty GFP vector (control), GFP-H2AX or GFP-H2AX-S139A for 24 h. (B,C) Immunoblot analysis of soluble nuclear (S3) and chromatin bound (P3) protein fractions obtained from U-2 OS cells transiently (B; 24 h) or stably (C) transfected with empty GFP vector, GFP-H2AX or GFP-H2AX-S139A and treated with 10 μM aphidicolin (APH) for 1 h to block DNA replication. Staining for GFP detects expression of tagged proteins, whereas staining for H2AX detects endogenous protein. Immunoblot for ORC2 is shown to rule out carry-over between fractions. Ponceau S staining demonstrates protein loading.