Figure 2

NSPCs constitutively release angiogenesis activators and induce upregulation of the mTOR and TGF-β signaling pathway components in ECs. A: Pre-conditioned medium collected from the NSPCs cultured for 24 hours, was analyzed using a Mouse Angiogenesis Antibody Array Kit. The array membrane with the immobilized specific antibodies against different pro-angiogenic factors, was incubated with the pre-conditioned medium. The bound proteins were visualized with secondary detection antibody followed by chemiluminescence detection. B, graph: ECs were grown as monoculture (clear bars) or were co-cultured with NSPCs (black bars). After 24 hours in culture, the cell lysates were subjected to Western blot. Immunoblot analysis was performed using the antibodies against mTOR, Rictor, Raptor, SMAD-2/SMAD-3, and actin. Signal intensities were quantified with AlphaEaseFC densitometry software. Relative density was calculated by the ratio of the protein of interest vs actin band intensities, and displayed graphically. Values represent the mean ± S.E.M. (n = 3, p < 0.05, Student's t-test). Immunoblot on the right depicts the representative experiment. Lane 1: ECs grown as a monoculture; lane 2 - ECs grown in co-culture with NSPCs.