Goat anti rabbit or goat anti mouse horseradish peroxidase hrp conjugated secondary antibodies

AGS cells were transfected with miRNA mimics and controls for 48 h. Subsequently, protein lysates were harvested using RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1 % Triton X-100; and 0.1 % SDS) containing protease (1:100, Roche, USA) and phosphatase (1:100, Sigma-Aldrich, USA) inhibitors. The protein concentrations were determined using a bicinchoninic acid assay (Pierce, Thermo Scientific, USA). Sixty μg of the proteins were separated by SDS-PAGE and transferred (Bio-Rad, USA) to PVDF membranes (Millipore, USA). Human PI3K-β, TSC1 and mTOR protein expression levels were quantified using rabbit polyclonal antibodies specific for each protein (1:1000, Cell Signaling, USA). The expression levels of these three proteins were standardized to human α-actin using a mouse polyclonal anti-α-actin antibody (1:5000, Millipore, USA). Primary antibodies were detected using goat anti-rabbit or goat anti-mouse horse-radish peroxidase (HRP)-conjugated secondary antibodies (1 : 5000, Santa Cruz Biotechnology, USA). Immunoreactive bands were visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, USA) according to the manufacturer's instructions, and then quantified by densitometry using a ChemiGenius Gel Bio Imaging System (Syngene, USA).