Total s6 kinase

Total proteins were extracted through 30-minute incubation on ice with Nonidet P-40 lysis buffer containing protease and phosphatase inhibitors, and resolved on Bolt Bis-Tris Plus polyacrylamide gels (Life Technologies). Antibodies against PARP (cat# 9532S), Phospho-Akt (S473; cat# 4060S), Akt (cat# 4685S), Phospho-Erk (T202/Y204; cat# 9101S), Erk1/2 (cat# 9102S), Phospho-S6 ribosomal protein (S235/236; cat# 2211S), total S6 ribosomal protein (cat# 2317S), Phospho-S6 kinase (cat# 9234S), total S6-kinase (cat# 2708S), Tuberin/TSC2 (cat# 4308S), Phospho-RSK (S380) (cat# 9335), total RSK (cat# 9355), Fatty Acid Synthase (cat# 3180S), Acetyl-CoA Carboxylase (cat# 3676S), Stearoyl-CoA desaturase 1 (cat# 2794S), CCTα (cat# 6931S) and BrdU (5292S) were obtained from Cell Signaling Technology (Danvers, MA). Anti-beta actin antibody (cat# A5316) was obtained from Millipore Sigma (St. Louis, MO) and anti-CPT1A antibody (cat# ab128568) from Abcam (Cambridge, MA).

Total proteins were extracted through 15-minute incubation with Nonidet P-40 lysis buffer and resolved on Bolt Bis-Tris Plus polyacrylamide gels (Life Technologies). Phospho-S6 kinase (cat# 9234S), total S6-kinase (cat# 2708S), and Tuberin (cat# 4308S) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Beta-actin (cat# A5316) was obtained from Sigma (St. Louis, MO). Primary antibodies were diluted 1:1000 in 5% bovine serum albumin or 5% non-fat milk in Tris-buffered saline (TBS) with 0.5% Tween 20, following the manufacturer’s instructions, and probed on nitrocellulose membrane overnight at 4°C.