Morphine inhibits Wnt secretion. Wild-type HEK293 (A) and 293-MOR cells (B) were transfected with myc-tagged Wnt2 cDNA. 24 hours after transfection, cells were treated with either 10 μM morphine or 10 μM DAMGO for an additional 24 hours. In A and B, open bars represent non-drug-treated controls, black bars represent morphine-treated cells, and grey bars represent cells treated with DAMGO. (C) 293-D2R cells were transfected and processed as in A and B, but were cultured in the presence of 10 μM dopamine instead of morphine. Media was collected, proteins separated by SDS-PAGE, then transferred to a PVDF filter. Filters were probed with anti-myc antibodies. Open bars represent non-drug-treated controls, black bars represent drug-treated cells. (D) Overexpression of GPR177 rescues Wnt secretion. 293-MOR cells were transfected with myc-tagged Wnt2 and GPR177 cDNAs. Cells were processed and proteins visualized as described above. Open bars represent no drug treatment, black bars represent morphine-treated cells, and grey bars represent cells overexpressing GPR177. Bands were analyzed by densitometry and quantitated using ImageJ software. Data was analyzed using a two-sided Student's t-test (expressed as mean ± SEM; n= 4-5; *p < 0.05; **p < 0.01).