mTOR pathway cell lysis and immunoblotting.

Except where noted, adherent cells were washed once in ice cold PBS and lysed in 40 μL ice cold RIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% triton) plus protease/phosphatase inhibitor cocktail and 5 mM NaF. Lysate was removed from the well, added into 1.5 mL Eppendorf tubes, and sonicated with 10 one-second pulses at maximum amplitude with a Fisher Scientific Model 120 Sonic Dismembrator (Thermo Fisher). Lysates were centrifuged at 12,700 rpm speed at 4°C for 15–20 min, and the supernatant was isolated to exclude debris. Protein concentration was quantified using the Pierce Microplate BCA Assay Kit (Thermo Fisher). Samples were prepared with 10x Bolt Sample Reducing Agent (Life Technologies) and 4x Bolt LDS Sample Buffer and run on Bolt 4–12% Bis-Tris Plus gels at 100V for 1 h and 45 min using Bolt MES running buffer (Thermo Fisher) diluted to 1x. Protein was transferred to iBlot nitrocellulose membranes, blocked in Odyssey Blocking Buffer (Li-Cor) for 1 h at room temperature and incubated with primary antibodies. Primary antibodies used (dilution) were goat anti-actin (1:5000) from Santa Cruz, rabbit anti-phospho-Akt (Ser473) (1:1000), rabbit anti-Akt (1:1000), rabbit anti-RPS6 (1:1000 or 1:5000), rabbit anti-phospho-RPS6 Ser235/236 (1:1000 or 1:2000), rabbit anti-4EBP1 (1:1000), rabbit anti-phospho-4EBP1 Thr37/46 (1:1000), rabbit anti-ATF4 (1:1000) and rabbit anti-GAPDH (1:500) from Cell Signaling Technologies, and mouse anti-tubulin (1:10,000) from Fisher Scientific. Membranes were probed overnight at 4˚C or for 1 h at room temperature with rocking. Then, membranes were washed three times for 7 min in Tris-buffered saline (ISC BioExpress, Cat No. 0788) with 0.1% Tween-20 (TBST). Secondary antibodies used were IRDye 680RD Donkey anti-Mouse IgG, IRDye 680RD Donkey anti-Goat, and IRDye 800 Donkey anti-Rabbit, all from LI-COR. Samples were probed with secondary antibodies at 1:15,000 in Odyssey Blocking Buffer (LI-COR) diluted 1:1 with TBST for 1 h at room temperature. Membranes were washed three times for 7 min in TBST, then imaged with the LI-COR Odyssey CLx Imaging System.