Association of Cdc25 with 14-3-3 proteins before and
after DNA damage is not dependent on Chk1 or Cds1. Strains with
integrated cdc25:3HA were exposed to 40 μm
CPT for 2 hr. Lysates were prepared and subjected to
immunoprecipitation with antibody to Rad24 (UMDNJ-55) and Rad25
(UMDNJ-56). (A) Rad24 immunoprecipitates were separated by
SDS-PAGE and immunoblotted with antibody to detect Cdc25 (12CA5,
top) and Rad24 (UMDNJ-55, bottom). The samples in
lanes 1 and 2 are precipitates of wild-type cells
with preimmune sera. The samples in lanes 3–8 are
precipitates with Rad24 antisera (UMDNJ-55). (Lanes
3,4 Wild-type cells (BN71); (lanes
5,6) chk1Δ cells (NW291);
(lanes 7,8) chk1Δ
cds1Δ cells (NW373). (B) Rad25
immunoprecipitates were separated by SDS-PAGE and immunoblotted with
antibody to detect Cdc25 (12CA5, top) and Rad25 (UMDNJ-55,
bottom). The samples in lanes 1 and 2 are
precipitates of wild-type cells with preimmune sera. The samples in
lanes 3–8 are precipitates with Rad25 antisera (UMDNJ-56).
(Lanes 3,4) Wild-type cells (BN71); (lanes
5,6) chk1Δ cells (NW291);
(lanes 7,8) chk1Δ
cds1Δ cells (NW373). (C) Samples of the
lysates used for immunoprecipitation in A and B were
immunoblotted for Cdc25 protein with mAb12CA5. The amount of Cdc25 in
the lysates is increased no more than twofold by DNA damage (estimated
by Western blot analysis of serial dilutions of the samples from
wild-type cells).