Antibodies for β actin

Total cellular extracts were prepared using RIPA Lysis and Extraction Buffer containing three Protease Inhibitors (Santa Cruz, Dallas, TX, USA). Western blot analyses were performed as previously described.^{24 (link)} Antibodies for β-actin (1:2000) and GAPDH (1:5000), LC3A/B (1:1000), AKT1 (1:1000), p-AKT1 (1:1000), AKT (1:1000), p-AKT (1:1000), mTOR (1:1000), p-mTOR (1:1000) were purchased from Abcam and Cell Signaling Technology, respectively. SC79, an AKT activator was purchased from Sigma Aldrich. Blots were visualized using ECL chemiluminescence reagents from Thermo Fisher Scientific.

Antibodies for β-actin were obtained from Abcam, and anti-phosphorylated (p)NKCC2 was a gift from Dr. Kerim Mutig. Lipofectamine reagents, TRIzol, and SDS polyacrylamide bis-Tris gels were from Invitrogen. Silencing of TNF mRNA was accomplished using the lentiviral vector psiLv-U6 (GeneCopoeia) and the lentivirus purification kit from Takara Bio USA. Recombinant mouse TNF was obtained from PharMingen. Polyvinylidene difluoride membranes were obtained from Amersham. The Nonidet P-40 lysis buffer contained protease inhibitors (Roche Diagnostics) for anti-pNKCC2 analysis. Tissue culture media were obtained from Life Technologies (Grand Island, NY). The luciferase assay kit was from Promega (Madison, WI). All other chemical reagents were purchased from Sigma.