Fig 6. Only lipid phosphatase-active Pten restores normal pAkt levels at 4dpf.

At 4dpf, single embryos from a ptena+/-ptenb-/- incross were cut in half. The trunk region was used for genotyping and the anterior half was lysed and processed for immunoblotting. Lysates from siblings and ptena-/-ptenb-/- embryos were run side by side on gels and blotted. The membranes were probed with phosphospecific anti-pAkt antibody (directed against pSer473), and subsequently sequentially stripped and probed with Akt-specific antibody, Tubulin-specific antibody as a loading control and PTEN-specific antibody, which only detects endogenous Ptena. The elevated pAkt levels in pten double homozygous embryos (see non-injected control) could be reduced to physiological levels by microinjection of either Ptenb WT or Ptenb Y138L RNA, indicating that both constructs have similar lipid phosphatase activities.