Western lightning plus enhanced chemiluminescence ecl substrates

Radioimmunoprecipitation assay buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin) was added to samples for protein extraction. For Western blotting, 30 µg of the total lysates was separated using 6% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After blocking with dried nonfat milk for 1 h, the membrane was incubated overnight with primary antibodies at 1:3000 dilution. The primary antibodies and antibodies against phosphorylated epitopes used in this study were mTOR, phospho-mTOR (Ser2448), p70S6K, and phospho-p70S6K (all purchased from Cell Signaling Technologies, Danvers, MA, USA). β-actin (1:5000 dilution; Sigma Aldrich, St. Louis, MO, USA) was used as the internal control. Horseradish-peroxidase-conjugated goat anti-mouse IgG (Sigma Aldrich, St. Louis, MO, USA) and goat anti-rabbit Immunoglobulin G (IgG) (Sigma Aldrich, St. Louis, MO, USA) were used as secondary antibodies. Western Lightning^® Plus-Enhanced Chemiluminescence (ECL) Substrates (PerkinElmer, Inc., Boston, MA, USA) were used to visualize the proteins.

Lysis buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1% Nonidet P-40 (NP-40), 1 mM ethylene glycol tetra-acetic acid (EGTA), 1% sodium deoxycholate,1 mM β-glycerophosphate, 1mM Na3VO4, 2.5 mM sodium pyrophosphate, and 1 µg/mL leupeptin) was added to samples for protein extraction. For Western blot, 30 µg of the total lysates were separated using 6% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After blocking with dried nonfat milk for 1 h, the membrane w-as incubated overnight with primary antibodies at 1:3000 dilution. The primary antibodies and antibodies against phosphorylated epitopes used in this study were β-actin, cleaved caspase 3, p-p38 kinase, and p-ERK1/2 kinase (Thr202/Tyr204) (all purchased from Cell Signaling Technologies, Danvers, MA, USA). β-actin (1:5000 dilution; Sigma Aldrich, St. Louis, MO, USA) was used as the internal control. Horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma Aldrich, St. Louis, MO, USA) and goat anti-rabbit IgG (Sigma Aldrich, St.Louis, MO, USA) were used as secondary antibodies. Western Lightning® Plus-Enhanced Chemiluminescence (ECL) Substrates (PerkinElmer, Inc., Boston, MA, USA) were used to visualize the proteins.