Figure 5
Phosphorylation of Bad by Pim prevents Bcl-X L binding. A) GST-Bad was transfected into HeLa cells along with either empty pCMV5 vector or active (W) or kinase dead (D) FLAG-Pim-1, 2, or 3 expression constructs. 24 h after transfection cells were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and imuunoblotted for Bcl-XL associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-XL, total Bad or FLAG. B) GST-Bad, or Ser112Ala, Ser136Ala, Ser155Ala, Ser136Asp or Ser136Glu mutants of GST-Bad were transfected into HEK-293 cells along with either empty pCMV5 vector or a FLAG-Pim-1 expression constructs. 24 h after transfection cell were serum starved for 18 h and were then lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-XL associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-XL,, total Bad and FLAG. C) GST-Bad, or Ser136/155Ala, Ser112/155Ala or Ser112/136Ala mutants of GST-Bad were transfected into HEK-293 cells with either empty pCMV5 vector or FLAG-Pim-1 expression constructs. 24 h after transfection cells were serum starved for 18 h and lysed. GST pull downs were then performed on the extracts and immunoblotted for Bcl-XL associating with the GST-Bad. Extracts were also immunoblotted with antibodies against total Bcl-XL, total Bad or FLAG.