Figure 1. Phosphorylation of Mesangial Lysate by Recombinant Active Akt.

Candidate Akt substrates were identified by phosphorylation of mesangial lysate with recombinant active Akt in the presence of [γ-P³²] ATP. Proteins were separated by SDS-PAGE and phosphoproteins detected by autoradiography. Minimal endogenous kinase activity was observed in mesangial lysate incubated in the presence of [γ-P³²] ATP without exogenous kinase, while incubation of lysate with active recombinant Akt resulted in phosphorylation of twenty proteins. Phosphoproteins were identified by comparing autoradiographs with corresponding coomassie-stained gels. Five candidate Akt substrates identified by peptide mass fingerprinting of trypsin-digested phosphoproteins using MALDI-MS are labeled on the autoradiograph.