Total or phospho specific antibodies

The potency (IC₅₀) of E6201 was determined via 14-point drug dose response viability assays for E6201, trametinib, cobimetinib, and vemurafenib. E6201 was provided by Spirita Oncology, LLC, and trametinib, cobimetinib, and vemurafenib were purchased from Selleck Chemicals (Houston, TX). Drug sensitivity was conducted as follows: 2000 cells were plated per well in a 96 well plate, 24 h later, drug treatments or a vehicle control were added, in triplicate, using a 2-fold dilution, 14-point drug dose response curve. Four days later, cell viability was assessed using a Cell-Titer Glo viability assay. Subsequent IC50 analyses were performed with PRISM 6, using a 3-variable, Hill-slope model.
Downstream pathway activation was evaluated using Western blot analysis. Briefly, melanoma cell lines were plated at a density of 200,000 cells per well in a 6-well plate. The next day, cells were washed in PBS and switched to 0.2% FBS containing media with DMSO vehicle control or E6201 treatment at the IC50 concentration for each cell line. Lysates were collected 1 h or 24 h after treatment. 20 μg of total protein were analyzed by SDS-PAGE. Phosphorylated and total Akt and ERK1/2 proteins and total GAPDH proteins were evaluated using total or phospho-specific antibodies from Cell Signaling Technology (Danvers, MA).