Figure 3.

Phosphorylation of Bad by Pim kinases in HEK-293 cells. A) FLAG-Pim-1, Pim-2 or Pim-3 were expressed in HEK-293 cells by transient transfection. 24 h after transfection cells were serum starved for 18 h and then stimulated with either IGF (100 ng/ml 5 min), sorbitol (0.5 M for 30 min), AICar (2 mM for 1 hour), UV-C (200Jm2 followed by 1 hour incubation at 37°C), hydrogen peroxide (1 mM for 1 hour), PMA (400 ng/ml for 15 min), anisomycin (10 μg/ml for 1 hour) or left unstimulated. Cells were lysed and Pim kinase activity measured by immunoprecipitation assays as described in the methods. B) GST-Bad was transfected into HEK-293 cells along with either empty pCMV5 vector or FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and then left unstimulated or stimulated with either PMA (400 ng/ml for 15 min) or forskolin (20 μM for 30 min). Cells were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression). C) GST-Bad was transfected into HEK-293 cells with empty pCMV5 vector or active or kinase dead FLAG-Pim-1-3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed and extracts immunoblotted for Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad and FLAG (to monitor Pim-1, 2 or 3 expression).

Macdonald et al. BMC Cell Biology 2006 7:1   doi:10.1186/1471-2121-7-1
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