Phosphorylation assays. HEK-293T/17 cells were plated in 6-well plates (Corning) 20-24 h prior to transfection. Each well was transfected with 0.33µg of receptor plasmid or empty vector. After 36-48h, cells were serum-starved for 2h before drug treatment by replacing complete growth medium with DMEM. To initiate stimulation, half (1ml) of the DMEM was removed and slowly replaced with 1mL fresh DMEM, containing a 2X concentration of vehicle, prosaptide, or head activator (HA) peptide. Plates were carefully returned to a 37°C incubator for 10 min. Following stimulation, cells were rapidly harvested in Laemmli buffer. Samples were sonicated and loaded into 4-20% Tris-Glycine gels (Bio-Rad) for SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked via shaking at RT for 30 min in Odyssey blocking buffer and incubated overnight with shaking at 4 °C with mouse anti-phospho ERK (Santa Xxxx) and rabbit anti-total ERK (Cell Signaling Technologies) or rabbit anti-phospho AKT (Cell Signaling Technologies and mouse anti-total AKT (Cell Signaling Technologies) in antibody buffer (equal parts blocking buffer and PBS + 0.1% Tween-20). Membranes were then washed three times in a wash buffer (PBS with 0.1% Tween-20) and incubated with Alexa-fluor anti-mouse 700-nm conjugated secondary antibody (1:20,000; Invitrogen) and anti-rabbit 800-nm conjugated secondary antibody (1:20,000; Li-Cor) for 30 min in antibody buffer. Blots were washed three times and rinsed in PBS until they were visualized on an Odyssey Imaging System (Li-Cor). Protein quantification was done using densitometry, performed with ImageJ software
