Targeting metabolic reprogramming by influenza infection for therapeutic intervention Smallwood et. al
Figure 5. BEZ235 mechanism acquired via PI3K/mTOR pathway restriction on metabolism. NHBE cells were infected with CA04 for 17 hours at MOI 1 +/- BEZ235 1hr prior to infection. (A) NHBE were harvested at indicated times and lysates subjected to immunoblotting.
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Immunoblotting: Cell were trypsinized, rinsed, and pelleted. Cell pellets were homogenized on ice in dounce homgenizer in cold Tris-EDTA + 0.1% NP-40 lysis buffer. Bradford assay (Coomassie Plus Pierce (Rockland, IL)) was used to determine protein concentrations of fresh lysates. 15µg of lysates per sample were reduced with NuPAGE reducing agent, diluted into NuPAGE LDS sample buffer, and boiled for 5 min prior to loading in 4-12% NuPAGE Bis-Tris gel for separation by electrophoresis. All gel reagents were purchased from Invitrogen (Carlsbad, CA). Proteins were transferred to nitrocellulose membranes and blocked with 2% bovine serum albumin (Sigma) in PBS (pH 7.4). The membranes were incubated with the indicated primary antibodies overnight at 4 °C or for 1-2 hours at room temperature followed by washing and incubation with appropriate horseradish peroxidase (HRP) conjugated secondary antibody (see reagents). The protein bands were detected using ECL plus from Amersham (Piscataway, NJ) and/or SuperSignal West Femptomax sensitivity substrate from Pierce (Rockford, IL).