Phosphor ampkα thr172

For Western blotting of AMPK, mTOR, AKT (total and phosphorylated) and actin, training was performed as described in stimulation experiments. Adherent monocytes were trained in 24 wells plate. After training and the resting period, cells were lysed in 150μl lysis buffer. Equal amounts of protein were subjected to SDS-PAGE electrophoresis using 7.5% polyacrylamide gels. Primary antibodies (1:500 and 1:50 000 (actin)) in 5% (w/v) BSA/TBST (5% bovine serum albumin/TBST) were incubated overnight at 4°C. HRP-conjugated antirabbit antibody or HRP-conjugated anti-mouse at a dilution of 1:5000 in in 5% (w/v) BSA/TBST were used for 1 hour at room temperature. Quantitative assessment of band intensity was performed by Image Lab statistical software (Bio-Rad, CA, USA). Following antibodies were used: actin antibody (Sigma, A5441), mTOR antibody (Cell Signaling, #2972, Leiden, the Netherlands), phospho-mTOR antibody (Ser2448) (Cell Signaling, #2971), AMPKα antibody (Cell Signaling, #2532), phosphor-AMPKα (Thr172) (Cell Signaling, #2531), Akt antibody (Cell Signaling, #9272), phosphor-Akt (Ser473) (Cell Signaling, #9271). At least four different individual experiments were repeated for each Western blot experiment.