Figure 3

Stability of PBIP1 in S and M phases of the cell cycle. To examine the level of PBIP1 stability and to assess the degree of PBIP1 delocalization during the cell cycle, HeLa cells were arrested at the G1/S boundary by double thymidine block, and then treated as described below. Top panel, Cells were released from the G1/S block into fresh medium. Two hours after release (DT 2 h), cells were treated with cycloheximide (20 μM) to inhibit protein synthesis. Where indicated, cells were additionally treated with proteasome inhibitor MG132 (10 μM) to prevent PBIP1 degradation. Bottom panel, Cells were released from the G1/S block into nocodazole (200 ng/ml)-containing medium (DT → Noc) to trap the cells in prometaphase. Ten hours after release, cells were treated as described above. Top and Bottom panels, the same amount of total cellular lysates (50 μg) were treated with either 200 U of λ phosphatase for 1 h at 30°C or left untreated. Samples were mixed with SDS sample buffer, boiled, and separated by 10% SDS-PAGE. Anti-PBIP1 immunoblotting analysis was carried out at the same time for both the top and bottom samples to directly compare all the chemiluminascent signals. The same membrane was stained with Coomassie (CBB) for loading controls. Numbers at the Top and Bottom panels indicate the relative levels of PBIP1 comparison to that of the DT 2 h sample.