Mapping the Ku86 binding domain of vigilin. (A) Ku86 binds to the KH10–12 region of vigilin. HEK 293 cells were transfected with GFP-tagged vigilin mutants. Seventy-two hours after transfection, whole cell lysates were used for immunoprecipitation with anti-Ku antibody. The presence of vigilin mutants in the precipitates was detected by Western blotting with anti-GFP antibody. Vigilin KH1–7 (lane 7), KH8–14 (lane 8), and KH10–14 (lane 9) regions, but not KH13–14 (lane 10) regions associate strongly with RHA. (B) The association of KH10–14 with Ku86 is independent of RNA. Experiments were carried out as in A except that cell lysates were treated with 200 μg/mL RNase A at 4°C for 2 h before immunoprecipitation. After RNase treatment, vigilin KH10–14 still associates with Ku86 (lane 4).
