Figure 3

(a and b): Phosphorylation of GST fusion protein containing amino acids 1–173 of human EF1α by Akt. Phosphorylation of GST-EF1α by Akt 1 and Akt 2 is reported in panel a and b, respectively, in comparison with histone H2B and GST used as controls. 1.5 μg of each substrate was incubated with 100 ng of Akt isoforms for 45 min in the presence of 50 μM [γ³³P] ATP (specific activity, 4000 cpm/pmol) as detailed in Materials and Methods. Data are the mean of three independent experiments with SD indicated by vertical bars. (c and d): Phosphorylation of EF1α KAERERGITID peptide (EFtide) by Akt. 100 ng of active Akt 1 (panel c) or 2 (panel d) was incubated for the indicated time with peptide KAERERGITID (EFtide) or Akt substrate peptide RPRAATF (AKTide). Phosphorylation of EFtide (Star) and AKTide (Black dot) was performed at 1 mM and 25 μM concentration, respectively. The amount of ³³P incorporated was determined as described in the Materials and Methods. Data reported are the mean of three independent experiments.