Fig. 2

Akt is present at high levels in membrane rafts in osteogenically differentiating MSCs. A sucrose density flotation method was used to determine Akt localization to membrane rafts in MSCs cultured in either growth medium (GM) or osteogenic medium (OM). In this method, buoyant membrane rafts float to the upper fractions (4–7) of the 12-fraction sucrose gradient upon ultracentrifugation, separating from intracellular fractions that remain at the bottom of the gradient. Top panel: immunoblotting of the fractions for the Golgi marker GM130 indicates intracellular fractions (9–12). Middle panel: immunoblotting with a pan-Akt antibody shows that Akt was mostly located within intracellular fractions. However, significantly higher levels of Akt were detected in membrane rafts from cells cultured in OM on day 10. This is shown by overlapping distribution with caveolin-1 (Cav-1) in buoyant membrane rafts. Bottom panel: immunoblotting for Cav-1 indicates the presence of membrane rafts floating in the upper fractions. This result is representative of four different human bone marrow MSC donor sources tested. Molecular weight standards are shown to the left of the immunoblots