Novex 4 20 tris glycine miniprotein gels

Total cell lysates were prepared using the M-Per Protein Extraction Reagent (ThermoScientific, Rockford, IL, USA) according to manufacturer instructions. Protein concentration was measured with BCA assay (Thermo Scientific). Equal amounts of protein (60 μg) were heat-denatured in lane marker reducing sample buffer (Thermo Scientific), resolved by SDS-PAGE using Novex 4–20% Tris-Glycine MiniProtein Gels (Thermo Scientific), and transferred to PVDF membranes (Millipore,Darmstadt, Germany).The filters were blocked in 5% BSA for 1h at room temperature and then incubated overnight at 4°C with primary antibody directed against p70 S6 Kinase and phospho-p70 S6 Kinase(Thr389) (Cell Signaling rabbit mAb #2708, rabbit mAb #9234) and antibody against beta-tubulin (Millipore, #05-661). Peroxidase conjugated IgG (Santa Cruz, CA, USA) was used as secondary antibody. Membrane-bound immune complexes were detected by ultra-sensitive enhanced chemiluminescence system (Thermo Scientific) on a photon-sensitive film (Hyperfilm ECL, GE Healthcare, Milano, Italy). Quantification was performed by densitometric analysis using ImageJ64 software.

Proteins were extracted from cultured cells using Lysis Buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 1% IGEPAL CA-630; and 0.5% Triton X-100) supplemented with protease inhibitors at 4°C. Cell lysates were spun at 11,000g for 30 minutes at 4°C and the supernatants contained the detergent-soluble fraction. The pellets containing the detergent-insoluble fractions were resuspended using the Lysis Buffer. Both the detergent-soluble and -insoluble protein samples were size fractionated on Novex 4-20% Tris-Glycine Mini Protein Gels (Thermo Fisher Scientific: XP04200BOX) and transferred overnight onto Immuno-Blot PVDF membranes at 4°C. The membranes were blocked for 30 minutes at room temperature using 3% Blotting-Grade Blocker in 1X PBST, and incubated with indicated antibodies overnight with rotation/nutation at 4°C. The following day, the membranes were washed in 1X PBST, incubated with indicated secondary antibodies for 2 hours at room temperature and proteins were detected using ECL Western blotting detection reagents.