Figure 5. Evaluation of agonist-dependent phosphorylation of phosphorylation-deficient human sst₃ receptor mutants.

HEK293 cells stably expressing wild-type hsst₃, 317–370 S/T-A, 4S/T-A, or 371–418 S/T-A were treated with 1μM SS-14, octreotide, pasireotide, somatoprim, or L-796/778 for 10 minutes at room temperature. The levels of phosphorylated sst₃ receptors were then determined using the phosphosite-specific anti-pS337/pT341 or anti-pT348 antibodies. Blots were subsequently stripped and reprobed with the phosphorylation-independent anti-HA antibody to confirm equal loading of the gels (anti-HA). Blots shown are representative of 3 independent experiments. The positions of molecular mass markers are indicated on the left (in kDa). Dose-response curves of AtT-20 cells stably expressing wild-type hsst₃, 317–370 S/T-A, 371–418 S/T-A, or 4S/T-A stimulated with sst₃-selective agonist L-796/778. Cells were stimulated with L-796/778 at concentrations ranging from 0.1nM to 5μM to yield a dose-response curve. EC₅₀ values are given as mean ± SD.