Fig. 9.

Expression of ORF2p containing mutations in selected putative phosphorylation sites outside of the endonuclease domain. 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 EN-RT- is a non-functional protein containing mutations in the endonuclease (D205A) and reverse transcriptase (D702A) domains. Control lanes indicate cells transfected with an empty vector. Lysates were probed with polyclonal antibodies generated against the human L1 ORF2 protein. Bottom panel: Western blot quantitation. For each sample, the signal detected for ORF2p was normalized to the total protein load. These relative numbers were expressed as a proportion of the relative number detected from the functional ORF2p. Asterisk denotes a significant difference in the steady-state levels relative to the functional ORF2p (t-test, P ≤ 0.05)