Gsk3α

Cells were lysed with RIPA-buffer (Radioimmunoprecipitation assay buffer). Gel electrophoresis was performed under reducing conditions with acrylamide gel and proteins were transferred to a nitrocellulose membrane. As primary antibodies, GPRC5a (NOVUS, NBP1-89743, Littleton, CO, USA), phosphorylation -GSK-3β (Cell Signaling, #5558, Danvers, MA, USA ), GSK-3β (Cell Signaling, #12456), GSK-3α (Cell Signaling, #4337), β-Catenin (Cell Signaling, #8480), PPARγ (Cell Signaling, #2443), Erk1/2 (Cell Signaling, #4695), and phosphorylation Erk1/2 (Cell Signaling, #4370) were used for detection. GAPDH (Cell Signaling Technology, 5175S) served as loading control. HRP (Horseradish peroxidase) -linked anti-rabbit IgG (Immunoglobulin G) (Cell Signaling Technology, 7074) was used as the secondary antibody. Quantification of signals was performed by an Amersham Imager 600 (Pittsburgh, PA, USA) with SignalFire Elite ECL Reagent (Cell Signaling Technology, 12757S). All Western blot assays were carried out individually two times, which validates the reliability of the results.

For immunoblotting, about 50 µg of total protein from MDA-MB-231 or MDA-MB-468 cells were run in 12% SDS PAGE and transferred to PVDF membranes (GE Health Care, Piscataway, NY, USA) at 75 V for 2 h at 4 °C. The membranes were blocked in 5% non-fat dry milk-TBS-0.1% Tween 20 (TBST) at room temperature for 2 h and then incubated with primary antibody in the same buffer at 4 °C overnight. The following primary antibodies were used: antibodies against B2AR (1:1000, Abcam, Branford, CT, RRID: AB_2747383), MOR (1:1000, Cell Signaling, Danvers, MA, RRID: AB_177512) GSK3α (1:1000, Cell Signaling, Danvers, MA, RRID: RRID:AB_659836), GSK3β (1:1000, Cell Signaling, Danvers, MA, RRID: RRID:AB_10998934), and β-actin (1:10,000, Abcam, Branford, CT, RRID:AB_2737344). The membranes were washed for 10 min 6 times in TBST and then incubated with corresponding peroxidase conjugated secondary antibody (1:5000, Vector Labs, Burlingame, CA) at room temperature for 1 h. The membranes were washed in TBST for 10 min 6 times and incubated with ECL reagent (Thermo Fisher Scientific, and were developed on the film by autoradiography. The protein band intensities were determined by Image J analysis software (National Institutes of Health, Bethesda, MD) and normalized with corresponding β-actin band intensity.