Phospho raptor ser792

Protein extraction was performed using RIPA buffer in the presence of a complete Mini EDTA-free protease inhibitor (Roche) and PhosSTOPTM phosphatase inhibitor (Roche). Protein separation was performed by SDS-page using Bolt 4 to 12% Bis-Tris protein gels (Invitrogen). Transferences were done in an iBlot 2 gel transfer device using iBlot 2 transfer nitrocellulose stacks (Invitrogen). Membranes were blotted against phospho-AKT (Ser473) (Cell Signaling), phospho-RAPTOR (Ser792) (Cell Signaling), phospho-p70 S6 Kinase (Thr389) (Cell Signaling), β-ACTIN (Cell Signaling) and their correspondent HRP-conjugated secondary antibodies. The revelation was performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and images were acquired using an ImageQuant LAS 4000 (GE Healthcare).

Cell lysates were subjected to SDS-PAGE, followed by transfer to nitrocellulose membrane. The membranes were then probed with primary Abs against total BCMA (cat. no. 27724-1-AP; Proteintech), Pan-Akt (cat. no. 4691; Cell Signaling Technology), pAkt (cat. no. 4060; Cell Signaling Technology), phospho-p38 MAPK (cat. no 4511; Cell Signaling Technology), p38 MAPK (cat. no. 8690; Cell Signaling Technology), phospho-mTOR ser2448 (cat. no. 5536; Cell Signaling Technology), total mTOR (cat. no. 2983; Cell Signaling Technology), Raptor (cat. no. 2280; Cell Signaling Technology), Phospho-Raptor Ser792 (cat. no. 89146; Cell Signaling Technology), phospho-p70 S6K (cat. no. 9204; Cell Signaling Technology), phospho-S6 (cat. no. 9204; Cell Signaling Technology), ATMINASCIZ (cat. no. AB3271-I; Millipore Sigma), and β-actin (cat. no. sc-47778 HRP; Santa Cruz Biotechnology) at 4°C overnight. The blots were then washed and probed with HRP-conjugated anti-goat (cat. no. sc-2020; Santa Cruz Biotechnology) or HRP-conjugated anti-rabbit (cat. no. A16110; Thermo Fisher Scientific) as appropriate. The blots were developed with Bio-Rad Western C Developing Reagent (cat. no. 170-5060; Bio-Rad) and visualized with a Chemidoc digital imager (cat. no. 1708280; Bio-Rad).

Antibodies directed against AMPKα1/α2 (#2603), ACC (#3676), phospho-ACC-Ser79 (#3661), PSD-95 (#2507), GluA1 (#13185), Raptor (#2280), phospho-Raptor-Ser792 (#2083), ULK1 (#8054), phospho-ULK1-Ser555 (#5869), SQSTM1/p62 (#5114), and LC3B (#2775) were obtained from Cell Signaling Technology. Anti-AMPKα2 (AF2850) was from R&D Systems. Anti-phospho-AMPK-Thr172 (sc-33524), synapsin Ia/IIb (sc-376622), Homer 1bc (sc-25271), synaptophysin (SYP) (sc-17750), GluN1 (NMDA zeta1; sc-1467), and SNAP25 (sc-376713) were from Santa Cruz Biotechnology. Anti-actin (#612656) and Munc (#610336) antibodies were from BD Transduction Laboratory. MAP2 (#M1406) antibody was from Sigma. SMI-312 (#837904) antibody was from Biolegend. AICAR and MRT68921 were purchased from Tocris, GSK621 was from Selleckchem, and Bafilomycin A1 was from Cell Signaling.