Protein extraction and immunoblotting

For protein extraction, cultured cells were lysed in RIPA lysis buffer (Cell Signaling, Danvers, MA) with freshly added PMSF, sodium orthovanadate and complete protease inhibitors [23 (link)]. Protein concentration was determined with the Pierce^TM BCA protein assay (ThermoFisher, Philadelphia, PA). Immunoblotting was done with anti-IGF2 antibody (Biorbyt, Cambridge, UK) according to the manufacturer’s protocol. Briefly, 4–12% Bis-Tris polyacrylamide gels (NuPage, Invitrogen) were equally loaded with 5–30μg of protein, electrophoresed at 140-200V, and transferred to nitrocellulose membrane by iBlot dry transfer. Membranes were blocked with either 5% BSA or LI-COR TBS blocking buffer for 30 minutes to 1 hr. The membrane was probed with primary antibodies to IGF2 (Biorbyt, Cambridge, UK) and pSTAT3, STAT3, TSC2, pS6, S6, GAPDH and β-actin (all from Cell Signaling Technology, Inc., Beverly, MA) at 4°C overnight. Appropriate secondary antibodies were incubated for 1 hr at room temperature. Blots were developed either by ECL prime Western blotting detection reagents (GE—Amersham Bioscience, Piscataway, NJ) or by secondary fluorescent antibodies (LI-COR, Lincoln, NE). Images were captured using a LI-COR Odyssey-Fc imager with LI-COR advanced imaging software.