Fig. 2.

Coilin directly interacts with Ku70 and Ku80. GST-pulldown of Ku80 and Ku70 by GST-fused full-length coilin or mutants thereof. (A) Bacterially purified GST or GST fused to various coilin fragments was incubated with bacterially purified soluble Ku80, followed by extensive washing of the beads, SDS-PAGE, and western transfer. The blots were probed separately with anti-Ku80 and anti-GST antibodies. The input lane accounts for 10% of Ku80 used in the pulldown reactions. (B) Soluble, bacterially expressed T7-tagged Ku70 was incubated with GST and GST-coilin fusion proteins and treated as described above. The blot was probed with antibodies to the T7 tag to detect Ku70, and GST antibodies to monitor the level of GST fusion protein used in each reaction. (C) Schematic representation of the different coilin fragments that were fused to GST and used in the pulldown reactions. The coilin self-interaction domain and RG box, which mediates coilin interaction with SMN, are indicated. (D) The C-terminus of Ku80 is not necessary for coilin interaction. Soluble full length T7-tagged Ku80 (N-732aa) and an N-terminal T7-tagged Ku80 (N-565aa) fragment were incubated with GST (lanes 3 and 4) or GST-coilin (lanes 5 and 6) beads. The washed beads were subjected to SDS-PAGE, western blotted and probed with T7 antibodies to detect Ku80 or the N-565 fragment. Ponceau S (bottom panels) was used to detect the amount of GST proteins in the reactions, and the input lanes account for 10% of the soluble protein used in the pulldown reactions.