Fig. 7.

Expression of EN proteins containing mutations in putative phosphorylation sites can induce DNA damage. a Representative western blot analysis of total cell lysates harvested from HeLa cells transiently transfected with the indicated EN putative phosphorylation mutant plasmids. EN is the functional protein and EN- is a non-functional protein containing inactivating mutations (D205A/H230A). Control lanes indicate cells transfected with an empty vector. a Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 endonuclease domain [42, 43], top panel; anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, middle panel; and anti-GAPDH to serve as a loading control, bottom panel. b Western blot quantitation. For each sample, the signal detected for ENp was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp. Asterisk denotes a significant difference in the steady-state levels relative to the functional ENp (t-test, P ≤ 0.05). c Western blot quantitation. For each sample, the signal detected for γH2AX was normalized to the signal detected for GAPDH. These relative numbers were expressed as a proportion of the relative number detected from the functional ENp