SH-SY5Y cells were incubated at 37°C for 30 minutes with vehicle control (0 µM) and varied concentrations of AM404 (ab120095). Increased expression of c-Fos in SH-SY5Y cells correlates with an increase in AM404 concentration, as described in literature.Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg 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 ab7963 at 1 µg/ml and 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.