Bms 754807

The following inhibitors were used in this study: IGF-1R inhibitor (BMS-754807; Funakoshi Co. Ltd., Tokyo, Japan), PI3K inhibitor (LY294002; Wako Pure Chemical Co.), AKT inhibitor (GDC-0068; Funakoshi Co. Ltd.), mTOR inhibitor (Rapamycin, Funakoshi Co. Ltd.), ERK inhibitor (U0126; Cosmo Bio Co. Ltd.), and JAK inhibitor (InSolution^TM JAK inhibitor I, Calbiochem, San Diego, CA, USA).
The right and left halves of brain slices were used. In ‘the control group’, the slice was incubated in the CM containing solvent (ethanol, DMSO, or saline) at the same concentration as that used to dissolve the respective inhibitor and 11-KT. In ‘the inhibitor group’, the slice was incubated in CM containing one of the inhibitors and the solvent to dissolve 11-KT. In ‘the 11-KT group’, the slice was incubated with 10 nM 11-KT, and the solvent was added at the same concentration as that used to dissolve the respective inhibitor. In ‘the 11-KT + inhibitor group’, the slices were incubated in CM containing 10 nM 11-KT and one of the inhibitors.
The slices for ‘control’ and ‘inhibitor’ groups originated from the same animal. Similarly, the slices for ‘11-KT’ and ‘11-KT + inhibitor’ groups originated from the same animal. We used 10–14 animals for the two pairs of the following four groups: the ‘control’, ‘inhibitor’, ‘11-KT’, and ‘11-KT- and inhibitor’ groups.

The antibodies and reagents used in this study were obtained from the following sources. Anti-IRS-1 antibody and anti-IRS-2 antibody for immunoprecipitation were raised in rabbits as described previously [47 (link)]. Anti-IRS-2 antibody for immunoblotting, anti-IGF-IR β antibody, anti-HSP90 α/β antibody and anti-ubiquitin antibody (P4D1) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-USP9X antibody was kindly provided by Dr. Stephen A. Wood (Griffith University, Queensland, Australia). Anti-USP9X antibody was also obtained from Abcam (Cambridge, MA, USA). Anti-FLAG antibody and anti-phosphotyrosine antibody (4G10) were from Sigma (St. Louis, MO, USA). Anti-Myc antibody (9E10) was from Millipore (Billerica, MA, USA). Anti-Akt antibody, anti-pAkt (Ser473) antibody, anti-Erk1/2 antibody, anti-pErk1/2 antibody and anti-IGF-IR β antibody were from Cell Signaling Technology (Dancers, MA, USA). MG132 was from Cell Signaling Technology (Dancers, MA, USA). Cycloheximide was from Nacalai Tesque (Tokyo, Japan). PD98059 was from Sigma (St. Louis, MO, USA), BMS754807 was from Funakoshi (Tokyo, Japan). WP1130 was from Life Sensors (Malvern, PA, USA). Recombinant human IGF-I was kindly gifted by Dr Toshiaki Ohkuma (Fujisawa Pharmaceutical Co., Osaka, current Astellas Pharma Inc., Tokyo, Japan).