Fig. 3

Identification of potential targets of cobimetinib activity on NB cells. Antibody arrays to a panel of signaling molecules were used to screen for dephosphorylation effects by cobimetinib. Proteins were extracted from IMR-32 cells treated with 1 μM of cobimetinib and cell lysates were made with lysis buffer containing protease and phosphatase inhibitors. DMSO treated cells were used as control. Antibody arrays were incubated with 200 μg of proteins over night at 4 °C with gentle mixing and probed with HRPO conjugated anti-phospho Tyr/Ser/Thr antibodies as per manufacturer’s protocol. Presented is the map of the orientation of the antibodies on the array (a), luminographically developed blots of cobimetinib treated and DMSO treated blots (b) and the waterfall plot showing changes in phosphorylation status of each signaling molecule on the arrays (c). Findings were representative of two completely separate experiments