(A) Expression of TdT in Sf9 cells. Sf9 cells were
infected with recombinant baculovirus expressing TdT (lane 2); TdT and
His-Ku70 (lane 3); TdT and His-Ku86 (lane 4); TdT and
His-Ku70/86 (lane 5); and TdT and adenosine deaminase (ADA; lane
6). The baculovirus constructs used for infections are indicated above
the lanes. Lane 1 contains 30 ng of purified TdT. Extracts were made
72 h after infection, electrophoresed on SDS/10% PAGE, and
transferred to a PVDF membrane. The blot was probed with rabbit
anti-TdT polyclonal antibody at a 1:1,000 dilution and developed by
using the enhanced chemiluminescence method. (B)
Coimmunoprecipitation of Ku subunits with TdT. Approximately 50 μg of
extract obtained from single, double, or triple coinfections was
incubated with the anti-TdT 133D2 (lane 2), anti-polyHis (lanes 3–5),
and anti-ADA N1D1 (lane 6) mouse mAbs. The immunoprecipitated complexes
were resolved by SDS/10% PAGE, transferred to PVDF, and probed
with rabbit anti-TdT polyclonal antibody (1:1,000 dilution). The blots
were developed as described in A.