Figure 4

Ser136 is a critical site to allow phosphorylation of Bad by Pim in cells. A) 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 cells were serum starved for 18 h and were then lysed and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. B) 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 and extracts immunoblotted with antibodies against Bad phosphorylated on Ser112, Ser136 or Ser155, total Bad or FLAG. C) 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 cells were serum starved for 18 h and were then lysed and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. D) 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 and extracts immunoblotted for total Bad or FLAG (to monitor expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. E) GST-Bad was transfected into HEK-293 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 and extracts immunoblotted for total Bad or FLAG (to monitor Pim-1, 2 or 3 expression). Extracts were also run on SDS polyacrylamide gels, transferred to nitrocellulose and 14-3-3 overlays performed as described in the methods. F) As E except that GST pull downs were performed on the extracts and immunoblotted for 14-3-3 as described in the methods.