Additional file 7: Figure S4. of The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations

Western blot analysis of ORF2 proteins containing mutations in putative phosphorylation sites. Top panel: Representative western blot analysis of total cell lysates harvested from HeLa cells transfected with the indicated ORF2 putative phosphorylation mutant constructs. ORF2 is the functional protein and ORF2 RT- is a non-functional protein containing a mutation in the reverse transcriptase (D702A) domain. Control lanes indicate cells transfected with an empty vector. Lysates were probed with anti-γH2AX antibodies to detect the phosphorylation of histone H2AX in response to DNA damage, top; and anti-GAPDH to serve as a loading control, bottom. Bottom panel: 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 ORF2p. (PNG 263 kb)