Radiolabeled atp

HEK23T cells were transfected with Myc-mTOR, HA-Raptor or HA-Rictor using X-Treme gene transfection reagent (Roche). Two days after transfection, cells were lysed and incubated with the HA-antibody or Myc-antibody conjugated agarose beads for 1 hour. The beads were then washed three times with Lysis Buffer (without phosphatase inhibitors) and then in kinase assay buffer (1 mM beta-glycerolphosphate, 20 mM Tris pH 7.4, 12 mM MgCl₂ and 100 μM ATP). The radiolabeled ATP was from Perkin Elmer and the unlabeled ATP was from Sigma Aldrich. The beads were then incubated with the TBK1, ATP or the appropriate negative control at 30 degrees for 30 minutes. The reaction was stopped using SDS-PAGE Laemmli buffer. His-tagged recombinant 4EBP1 was purchased from Sigma-Aldrich.

Trypsin-EDTA (Cat# 25-053-CI), PBS (Cat# 21-031-CV), and laminin (Cat# 354232) were purchased from Corning. Recombinant EGF was purchased from Life Technologies (Cat# PHG0311). IGF was purchased from Sigma-Aldrich (Cat# I3769). PIM447 was purchased from Selleck Chemicals (Cat# S7985). AZD1208 was acquired from AdooQ Bioscience (Cat# A13203-750). DMSO was purchased from Thermo Fisher Scientific (Cat# 97064-724). Cycloheximide was purchased from VWR (Cat# 97064-724), and doxycycline was purchased from Sigma-Aldrich (Cat# D9891-5G). Recombinant myelin basic protein (MBP) was a gift from Dr. Greg Rogers, and recombinant ABI2 protein was purchased from Origene (Cat# TP300637). Radio-labeled ATP was purchased from Perkin Elmer (Cat# BLU502A). The antibody to ABI2 was purchased from Bethyl Laboratories (Cat# A302-499A-M). GFP (Cat# 2956S), HA (Cat# 3724S), HIF-1a (Cat# 14179S), p-IRS1 [S1101] (Cat# 2385S), PIM1 (Cat# 3247S), and WAVE2 (Cat# 3659S) antibodies were purchased from Cell Signaling Technology. The antibody for actin was purchased from BD Biosciences (Cat# 61656). PIM1 (Cat# ab75776) and Ki67 (Cat#ab833) antibodies used for immunohistochemistry were purchased from Abcam.