Physical association between GANP and MCM3.

(A) Association of MCM3 and GANP in cells in vivo. COS7 cells were transfected with complementary DNA (cDNA) constructs of both HA-MCM3 and FLAG-ganp. The NP-40 cell lysates were immunoprecipitated with anti-HA Ab, and Western blot analysis was carried out with anti-FLAG mAb to detect GANP. The transfectants with the single cDNA constructs were used as the negative control. (B) The cell lysate from WEHI-231 was immunoprecipitated with anti-GST, anti-GANP 42-23, or anti-MCM3 Ab. After separation by SDS-PAGE, the proteins were electrophoretically transferred to a membrane and probed with anti-MCM3 Ab. (C, left panel) In vitro kinase reactions were carried out with the anti-GANP immunoprecipitates washed with either NP-40 washing buffer or radioimmunoprotein-assay (RIPA) buffer. After the kinase reaction, the phosphorylated sample was washed further with the RIPA buffer and reimmunoprecipitated. The anti-GST immunoprecipitate was used as the negative control. (C, middle panel) Anti-GST, anti-GANP 42-23, and anti-MCM3 immunoprecipitates from WEHI-231 cell lysates were subjected to in vitro kinase assay. Normal rabbit serum was used as the control for anti-MCM3 Ab. The samples were separated by 7% SDS-PAGE. The bands corresponding to the sizes of GANP and MCM3 are indicated by arrows. (C, right panel) V8 cleavage mapping of 210-kd bands showed an identical cleavage pattern. As the control, an irrelevant V8-digested protein was separated in parallel. (D) Double staining with anti-MCM3 Ab and anti-CR1 mAb, or with PNA, was performed. The expression of MCM3 was up-regulated in the GC area.