Spinophilin

Total proteins from stably transfected MCF-7 and SUM159 cells were extracted with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate and 1% Nonidet P40). 25 μg of total cellular proteins were resuspended in laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCl, pH approx. 6.8) and heated at 95 °C for 5 minutes. Proteins were separated by a 4–15% Mini-PROTEAN^® TGX^™ Precast Gel (Biorad, Hercules, CA) and transferred onto a nitrocellulose membrane (Applichem, St. Louis, MO). The membrane was blocked for 1 hour with 3% non-fat dry milk in Tris buffered Saline/0.1% Tween-20. Immunoblotting was performed and antibodies specific for spinophilin (Cell Signaling, Danvers, MA, Cat.No. 9061S), the apoptosis marker PARP (Cell Signaling, Cat.No. 9542), pRb (directed against phosphorylated serine 807/8, Cell Signaling, diluted 1:1000 in 1% non-fat dry milk in Tris buffered Saline/0.1% Tween-20), and β-Actin (Sigma, Cat.No. A5441, clone AC-15) were detected using HRP-conjugated anti-mouse or anti-rabbit antibodies, respectively (Dako, Glostrup, Denmark). Visualization was performed using an enhanced chemoluminescence detection system (Super Signal West Pico, Thermo Scientific, Rockford, IL).

Western blots materials and reagents were purchased from Bio-Rad. Rabbit anti–phospho-p44/42 ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-GSK3β (Ser9), NMDA receptor 2A, NMDA receptor 2B, PSD95, spinophilin, tyrosine hydroxylase, phospho-Akt, mTOR, phospho mTOR, and mouse anti-GSK3β, Akt antibodies were purchased from Cell Signaling Technology. Rabbit anti-phospho tyrosine hydroxylase Ser40 was purchased from Phosphosolutions. Rabbit anti-Dopamine receptor 1, Dopamine receptor 2, c-Fos were obtained from Abcam. Rabbit anti–GAPDH (glyceraldehyde- 3-phosphate dehydrogenase) was purchased from Santa Cruz Biotechnology and rabbit anti-mGluR5 was obtained from Milipore Sigma. Antibodies validation data are available from their respective companies. For RNA extraction and qPCR Sigma-Aldrich DNase I, TRIzol reagent from ThermoFisher Scientific, iScript™ cDNA synthesis kit from Bio-Rad and Luna Universal qPCR Master Mix from New England Biolabs were used. Vector Elite ABC HRP kit (rabbit) used in immunohistochemistry was purchased from Vector Laboratories.