Protein was isolated with M-PER lysing buffer (Pierce, Rockford, IL, USA) and Western blot was done with anti-GAPDH rabbit mAb (Cell Signaling), anti-phospho-Akt (Ser473) rabbit mAb (Cell Signaling, Danvers, MA, USA), as described previously [22 (link), 23 (link)]. ELISA for VEGF (R&D Systems) in media collected from the transfected cells was performed according to the vendor's protocol.
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