Turbo transfer buffer

Cells were plated at 100,000 cells/well in 6-well plates and were allowed to adhere overnight prior to treating with BKM120 in DMEM with 10% FBS for 30 minutes, 1, 3, 24 or 48 hours. DMSO was used as vehicle control. Cells were lysed with RIPA lysis buffer (Cell Signaling, Danvers, MA), supplemented with a complete protease inhibitor (Thermo Fisher Scientific Inc., Waltham, MA). Cell lysates were collected and frozen at -80°C overnight to ensure complete cell lysis. Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Lysates were electrophoresed on a 7.5% or 12% SDS-polyacrylamide gel in running buffer (Bio-Rad). Thirty micrograms of protein was loaded per lane. Gels were semi-dry transferred to nitrocellulose membranes using the Turbo Transfer system with Turbo Transfer buffer (Bio-Rad). Primary antibodies used were: rabbit polyclonal cleaved caspase 3 and caspase 3, rabbit polyclonal Phospho-AKT (Ser473), rabbit polyclonal AKT, rabbit monoclonal Phospho-S6 Ribosomal Protein (Ser235/236), rabbit monoclonal S6 Robosomal Protein, mouse monoclonal mTOR and rabbit monoclonal Phospho-mTOR, rabbit polyclonal β-actin, and rabbit polyclonal Vinculin (Cell Signaling). Goat anti-mouse or anti-rabbit HRP secondary antibody were used (Cell Signaling). Protein bands were detected using Clarity Western ECL Substrate (Bio-Rad).

Cells were plated at 100,000 cells/well in 6-well plates and were allowed to adhere overnight prior to treating with 5 mM DFMO in RPMI-1640 with 10% FBS for 48, 72, or 96 hours. Drug supplemented medium was refreshed after 48 hours for 72 and 96 hours timepoints. H₂O was used as vehicle control. Cells were lysed with RIPA lysis buffer (Cell Signaling, Danvers, MA), supplemented with a complete protease inhibitor (Roche, Madison, WI). Cell lysates were collected and frozen at −80°C overnight to ensure complete cell lysis. Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Lysates were electrophoresed on a 12% SDS-polyacrylamide gel in running buffer (Bio-Rad). Thirty μg of protein was loaded per lane. Gels were semi-dry transferred to nitrocellulose membranes using the Turbo Transfer system with Turbo Transfer buffer (Bio-Rad). Primary antibodies used were: rabbit polyclonal LIN28B, rabbit polyclonal MYCN, and rabbit polyclonal β-actin (Cell Signaling). Goat anti-rabbit secondary antibody was used (Licor). Protein bands were detected by fluorescence using the Odyssey infrared imaging system (Licor).