PKB/Akt activity is constitutive in HCD57-SREI cells but activity is induced by EPO and SCF in HCD57 cells. (Top, A) HCD57 cells deprived of EPO overnight (lanes 1, 2, 5, 6) or HCD57-SREI cells cultured without EPO (lanes 3, 4, 7, 8) were left untreated (1, 3, 5, 7) or treated with 10 U EPO/mL for 5 minutes (lanes 6, 8) or 100 ng SCF/mL for 5 minutes (lanes 2, 4). Cell lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. The blot was probed with anti-phospho-PKB/Akt antibody (top panel). After being stripped, the blot was reprobed with a general anti-PKB/Akt antibody that recognizes all forms of the protein (bottom panel). (B) Time course of EPO and SCF-induced PKB/Akt activation. HCD57 cells deprived of EPO overnight were left untreated (lanes 1, 5) or treated with 10 U EPO/mL (lanes 2 through 4) or 100 ng SCF/mL (lanes 6 through 8) for indicated time periods. Cell lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. The blot was probed with anti-phospho-PKB/Akt antibody (top panel). After being stripped, the blot was reprobed with the general anti-PKB/Akt antibody used above (bottom panel). (C) PI3-kinase inhibitor blocked EPO-induced PKB/Akt phosphorylation in HCD57-SREI cells. HCD57-SREI cells cultured without EPO were left untreated (lanes 1, 7), pretreated with DMSO (lanes 2, 8), or pretreated with LY294002 (lanes 3 through 6, 9 through 12) at the indicated concentration for 4 hours. Then cells were left untreated (lanes 1 through 6) or treated with 10 U EPO/mL for 5 minutes (lanes 7 through 12). Cell lysates were prepared and subjected to Western blot analysis as described in Materials and Methods. The blot was probed with anti-phospho-PKB/Akt antibody (top panel). The blot was stripped of the phospho-specific antibody and reprobed with an antibody that recognizes both phosphorylated and nonphosphophorylated PKB/Akt to verify equal loading (bottom panel).