Figure 2

Expression and activity of HO-fusion protein in permissive cell system (293 T cells). (A) Depicts immunoblot of HO fusion protein upon infection with adeno-HO virus at different time points in 293 T-HO-CAT cells. A map of the virus showing the HO fusion is presented in [32], and generates a presumed chimeric protein with the 11.6 k adenovirus gene product. Cells were lysed using RIPA lysis buffer and equal amounts of proteins were loaded and run in a 10% SDS-PAGE gel and subsequently probed with anti-E3-11.6 K (fusion protein with HO) antibody. The blot in panel (A) was re-probed with phospho-(S/T) Q-ATM/ATR substrate antibody, and the region of the blot corresponding to the position of HO in panel (A) is shown. Also shown, the p(S/T)-Q banding pattern in 293 T cells which lack the episomes after Ad-HO infection, to serve as a control. ? denotes undetermined bands obtained after probing with p(S/T)-Q antibody. (B) HO activity was measured in an in vitro cleavage reaction. Cell extracts were prepared from 293 T cells (lanes 1–5) and cells carrying pRep4-HO-CAT episomes (T-HO-CAT cells) (lanes 6–7) after Ad-HO infection at given time points. The plasmid pMat-Puro, harboring the HO recognition site [16], was added exogenously at 250 ng per reaction. The plasmid was treated with cell extracts obtained from different time points from both 293 T cells and T-HO-CAT cells. HO activity was judged by presence/absence of a cleaved product. In reactions shown in lanes 1–7, we have added Xmn1 to produce the ~750 bp band and corroborate the cleavage at the HO site. Lane 8 shows the treatment of the pMat-Puro plasmid with extracts obtained from uninfected cells (Unc). “M” denotes the Promega 1 Kb DNA ladder as a molecular weight marker.