Figure 4
FACT-associated MCM4 undergoes a cell cycle-dependent phosphorylation change. (A) HeLa cells were immunoprecipitated with mAb 8D2 (lanes 1 and 2) and 10D1 (lanes 3-6). Immunoprecipitates were further subjected to buffer (lanes 1, 3, and 5) or alkaline phosphatase treatment (lanes 2, 4, and 6). The presence of MCM proteins was detected by Western blot analysis using anti-pan MCM (lanes 1-4) and α MCM4 (lanes 5 and 6) antibodies. The positions of MCM 3/4/6 are distinguished by arrowheads. The smear of immunoreactive signlas for MCM4 is also highlighted. (B) FACT-associated immunocomplex was isolated by α SSRP1 mAb 10D1 from HeLa cells at different cell cycle stages: exponentially growing (lane 1), G1/S phase (lane 2), S phase at 1 hr after double-thymidine block (lane 3), S phase at 3 hr (lane 4), and M phase (lane 5) and G2 (lane 6). The presence of MCMs and FACT heterodimers was detected by the specified antibodies. The top four panels are immunoblots for the immunoprecipitates, and the bottom two panels are for the lysate inputs. The two anti-Mcm4 blots are from different exposure lengths of signal development (longer exposure on top).