Figure 3 | Oncogene

Figure 3

From: BRCA1-mediated G2/M cell cycle arrest requires ERK1/2 kinase activation

Figure 3

The effects of BRCA1 and ERK1/2 kinases on the regulators of G2/M checkpoint. (a) MCF-7 cells were infected with Ad.Control (lanes 1–2) or Ad.BRCA1 (lanes 3–4) at 50 PFU/cell and incubated in medium containing either 50 μ M U0126 (lanes 2 and 4) or, as a control, 0.1% DMSO (lanes 1 and 3). After 24 h incubation, the cells were collected and analysed for Cdc2-Tyr15 phosphorylation (Cdc2-Tyr15) by immunoblotting using a phosphorylation-specific antibody. Total Cdc2 protein levels (Cdc2) in the lysates were measured by immunoblotting using an anti-Cdc2-specific antibody. (b) MCF-7 cells were treated as described above. Wee1 and Chk1 kinases were immunoprecipitated and assayed for kinase activities using GST-Cdc2 and GST-Cdc25C substrate, respectively (Wee1 activity and Chk1 activity) as described in Materials and methods. The amounts of Wee1 and Chk1 present in the immunoprecipitates (Wee1 IP Western and Chk1 IP western) and the levels of Wee1 and Chk1 protein in the cell lysates (Wee1 Western and Chk1 Western) were assessed by immunoblotting. Equal protein loading was confirmed by measuring Actin levels on the immunoblots using an anti-Actin antibody (Actin). (c) Cells were infected with Ad.BRCA1 (lanes 2–4) or Ad.Control (lanes 1 and 5–6) at 50 PFU/cell and cultured for 24 h in the presence of 100 μ M PD98059 (lanes 3 and 5), expression of dominant negative MEK1 (lanes 4 and 6), or as a control 0.1% DMSO (lanes 1 and 2). The resulting cell samples were analysed for kinase activities of Wee1 and Chk1 as described above (Wee1 Activity and Chk1 Activity). The amounts of Wee1 and Chk1 protein presented in the immunoprecipitates were determined by immunoblotting using specific antibodies (Wee1 IP Western and Chk1 IP Western)

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