Recombinant human insulin i2643

AP21967 (or its equivalent A/C dimerizer, 635057) was from Ariad Pharmaceuticals and Takara Bio Inc., respectively. GDC-0941 (G9252) was from LC Labs. Torin1 (10997) was from Cayman Chemicals. Rabbit Akt^pSer473 193H12 (4058), rabbit pan-Akt 11E7 (4685), rabbit S6K^pT389 108D2 (9234), rabbit mTOR 7C10 (2983), rabbit GAPDH D16H11 (5174), and rabbit γ-tubulin (5886) antibodies were from Cell Signaling Technology. Mouse RICTOR 7B3 (MA-5-15681) antibody was from Thermo Fisher Scientific. Mouse Sin1 1C7.2 (05-1044) antibody was from EMD Millipore. Mouse mCherry (632543) antibody was from Takara Bio Inc. GFP-Trap_A (gta-20), RFP-Trap_A (rta-10), and Atto488-conjugated GFP booster (gba-488) scAb were from ChromoTek. Recombinant human insulin (I2643) was from Sigma-Aldrich.
The constructs used in the study were generated using standard molecular biology protocols and will be deposited to the AddGene repository (72899–72909). pmCherry-PH^Akt was a gift from T.A. Leonard (Max F. Perutz Laboratories, Vienna, Austria), and pTagBFP-2×DHFR-CaaX was a gift from Y. Wu (Max Planck Institute for Molecular Physiology, Dortmund, Germany).

Autophagy modulating compounds including Latrepirdine, Rapamycin and SMER28 were used as controls in this study. Latrepirdine (Dimebolin dihydrochloride, molecular weight (MW): 392.37) was purchased from Biotrend AG, Zurich, Switzerland. Latrepirdine was dissolved into sterile double distilled water to 30 mM concentration. Rapamycin and SMER28 were purchased from Sigma (Missouri, USA) and solutions were prepared in DMSO (Dimethyl sulfoxide). Freshly prepared working stocks (1 mM) prepared from -80ºC frozen stocks were used for all experiments. Synthetic human Aβ₄₂ and Aβ₁₆ were purchased from ERI Laboratory (Connecticut). Recombinant human insulin (I2643, MW: 5800 Da) was purchased from Sigma.