Deptor

The neuronally differentiated SH-SY5Y cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min prior to washes in PBS and incubation with 10% BSA for 1 h. The cells were incubated for 1 h with DEPTOR (Millipore), mTOR (Cell Signalling Technology) and pan-neuronal marker (Millipore) antibodies at a 1:100 dilution in 1% BSA/PBS. The cells were then washed with PBS prior to a further incubation with secondary antibodies as previously described (21 (link)). Images were captured using a Plan Apo Neofluar 63X NA 1.25 oil objective (Zeiss) on a Zeiss Axiovert 200 M microscope and viewed using AxioVision software.

Reagents were obtained from the following sources: Pan-Tanc antibody has been previously described^{22 (link)} and Tanc2-specific antibody made in-house; rabbit polyclonal antibodies to Akt (#9272), phosphor-Akt (#9271, #4060), Erk1/2 (#9102), phospho-Erk1/2 (#9101), GSK3β (#9315), phospho-GSK3β (#9336), PTEN (#9559), phospho-PTEN (#9549), PI3K (#4257), phospho-PI3K (#4228), TSC1 (#6935), TSC2 (#4308), phospho-TSC2 (#3617), mTOR (#2983), mTOR (#2972, used for immunoprecipitation), phospho-mTOR (#2971), S6 (#2217), phospho-S6 (#4858), 4E-BP (#9644), phospho-4E-BP (#2855), S6K (#2708), phosphor-S6K (#9205), and PRAS40 (#2691) from Cell Signaling Technology; Raptor (#09–217), phospho-Raptor (#09–107), phosphor-PKCa (#07–790), NeuN (Abn90), and Deptor (#abs222) antibodies (Millipore); PKCa (#610108) antibodies (BD); Flag M2 (F1804), GFAP (G3893), and α-tubulin (T9026) antibodies (Sigma Aldrich); HA antibodies (M180-3) (MBL); GFP antibodies (sc-9996; Santa Cruz); HRP-labeled anti-mouse, anti-guineapig, and anti-rabbit secondary antibodies (Thermo Scientific); IRDye 800CW-conjugated and 680RD-conjugated goat anti-mouse and goat anti-rabbit antibodies (LI-COR Biosciences); Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen); CHAPS detergent (Thermo Scientific); rapamycin (LC Labs); ketamine (Yuhan Co).

Cells were harvested, lysed, and subjected to immunoblotting, as previously described^{32 (link)}. About 30–60 μg of protein were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, and the bands with appropriate exposure time are presented. Antibodies against the following proteins were used: DEPTOR (11816, for human samples; Cell Signaling Technology, Danvers, MA, USA; 1:1000), DEPTOR (09-463, for mouse samples; Millipore, Burlington, MA, USA; 1:1000), p53 (32532, for mouse samples; Cell Signaling Technology; 1:1000), p-AKT (S473) (4060; Cell Signaling Technology; 1:2000), t-AKT (4691; Cell Signaling Technology; 1:1000), p-S6K1 (T389) (9234; Cell Signaling Technology; 1:1000), PARP (9532; Cell Signaling Technology; 1:1000), caspase-3 (9665; Cell Signaling Technology; 1:1000), cyclin A2 (4656; Cell Signaling Technology; 1:1000), cyclin E2 (4132; Cell Signaling Technology; 1:1000), cyclin B1 (12231; Cell Signaling Technology; 1:1000), cyclin D1 (2978; Cell Signaling Technology; 1:1000), p-H3 (3377; Cell Signaling Technology; 1:1000), t-S6K1 (sc-230; Santa Cruz Biotechnology; 1:1000), NOXA (OP180; Calbiochem, San Diego, CA, USA; 1:1000), MDM2 (OP46; Calbiochem; 1:500), p53 (OP43, for human samples; Calbiochem; 1:1000), p21 (sc-6246; Santa Cruz Biotechnology; 1:500), actin (ET1701-80; HuaAn Biotechnology, Hangzhou, China; 1:10,000).