Evi-1 associates with CtBP1 through PLDLS.
(A) Evi-1 contains 2 putative CtBP-binding motifs. (B) The pME18S empty vector (lane 1), Evi-1 (lanes 2, 3), Δ544-607 (lane 4), Δ608-732 (lane 5), or Δ544-732 (lane 6) in pME18S was transfected into COS7 cells (2 × 106) with pRc/CMV empty vector (lanes 1, 2) or T7-CtBP in pRc/CMV (lanes 3 to 6). After 48 hours, cells were lysed and subjected to immunoprecipitation with anti-T7. T7-CtBP–bound Evi-1 was detected by Western blot by means of anti–Evi-1 (top). Positions of size markers (in kilodaltons) are indicated on the left. Expression of T7-CtBP and Evi-1 is monitored with anti–T7 (middle) and anti–Evi-1 (bottom), respectively. (C) Structures of the mutants of Evi-1. Mutations from DL to AS within the 5 amino acid motifs are underlined. (D) The pME18S empty vector (lane 1), Evi-1 (lanes 2, 3), AS/DL (lane 4), DL/AS (lane 5), or AS/AS (lane 6) was transfected into COS7 cells (2 ×106) together with pRc/CMV empty vector (lanes 1, 2) or T7-CtBP (lanes 3 to 6) in pRc/CMV. The immunoprecipitation was performed, and the results are shown as in Figure 2B.
