BT549 cells were incubated at 37°C for 1h with vehicle control (0 µM) and different concentrations of PD 173074 (ab141117) in DMSO. Decreased expression of RSK1 p90 (phospho T359 + S363) (ab32413) correlates with an increase in PD 173074 concentration, as described in literature.Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab32413 at 1/5000 dilutionand ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.