Figure 7

PGRMC1 influences phosphorylation of Akt after H₂O₂ treatment. The figure shows a Western blot of equal total protein amounts (20 μg/lane) from cell extracts of the indicated cells. Antibodies employed were specific for phosphorylated Akt (top panel); for the polypeptide backbone of Akt, actin; or for the HA-tag on the respective exogenously expressed PGRMC1 constructs, as indicated. Incubation with the respective antibodies was perfomed simultaneously for the lower panel. Previous experiments indicated no overlap of signal (data not shown). Akt is phosphorylated upon H₂O₂ exposure in susceptible MCF-7 cells stably transfected with wild-type PGRMC1 and the mutants indicated. In peroxide-resistant cells expressing the S56A/S180A mutant, Akt phosphorylation is always abrogated. In some experiments in which the Y179F/S180A mutant exhibited limited survival to peroxide (for example, Figure 6b this was also accompanied by lack of Akt phosphorylation (data not shown). These trends were also observed at a concentration of 100 μmol/l H₂O₂ with lower overall viability levels (data not shown). PGMRC, progesterone receptor membrane component; HA, hemaglutinin.