D1 Receptor Expression Is Regulated by MicroRNA with ethidium bromide. The captured images were inverted for visualization purposes. and used to generate a standard curve. The cAMP levels in each treated sample were assayed in triplicate. Western Blotting Tissues and cells were harvested and lysed, solubilized using the CellyticTM-M or MT reagent (Sigma) supplemented with 1 mM phenylmethylsulfonylfluoride (PMSF) and 1% protease and phosphatase inhibitor cocktails (Sigma). Protein amounts in the lysates were determined using the BCA assay (BioRad, Hercules, CA, USA) and equal amounts of total cell proteins (10 to 100 mg) were loaded on to 10% gels and separated using SDS-PAGE. Blots were developed using appropriate primary and secondary antibodies as described previously [8–10]. Total and phosphory- lated dopamine- and cAMP-regulated phosphoproteins (DARPP- 32) were detected using rabbit monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA). For detecting D1 receptor protein, we used a rat monoclonal anti D1 receptor antibody (Sigma) as described previously [8–10]. This antibody specifically detects the various glycosylated forms of D1 receptor protein (Figure S2). Equal loading in lanes was determined by post-hoc staining of the nitrocellulose blots with the amido black total protein stain. b-galactosidase Assay For the b-galactosidase assay, the CAD cell pellet was lysed for 10 minutes on ice in the b-gal lysis buffer (10 mM KCl, 1 mM MgSO4, 2.5 mM EDTA, 0.25% NP-40 detergent, 50 mM b- mercaptoethanol and 100 mM sodium phosphate buffer [pH7.2]). The lysate was spun at 14,0006g for 10 minutes and the protein amount in the supernatant measured using the Bradford protein assay (Pierce). The b-galactosidase reporter enzyme activity in the lysate was detected using chlorophenol red-b-galactoside (CPRG, Roche) as a substrate. The b-galactosidase enzyme cleaves the CPRG substrate to yield a colored product which has maximal absorbance at 575 nm and thus can be detected using a visible wavelength spectrophotometer. The b-galactosidase reporter enzyme activity was normalized to the total protein amount present in the lysate and further normalized for transfection efficiency. cAMP Measurements Cyclic AMP (cAMP) levels were measured using the Cyclic AMP XPH Assay Kit (Cell Signaling Technology). Briefly, CAD cells (106 cells) were plated and transfected with negative control or miRZipTM 142-3p constructs in 12-well plates (Costar, Corning, NY, USA) and cultured for 48 hrs. To minimize variations due to differences in cell plating density or growth rate, we used two wells for each treatment variable. CAD cells were treated for 10 minutes at 37uC with 0.3 mM IBMX (phosphodiestrase inhibitor), or 0.3 mM IBMX + increasing concentrations (0.03 mM to 10 mM) of D1 receptor agonist, A77636 (Sigma), and lysed in 12 well plates. Sample lysates from the two wells were pooled and 0.05 ml transferred into three independent wells of the 96-well EIA plate. The cAMP levels were measured by following the recommenda- tions of the kit manufacturer (Cell Signaling Technology). The final absorbance values were measured at 450 nm on a Model 680 Microplate Reader (BioRad). The amount of cAMP in each well was determined by comparing the absorbance values of the treated variables with a series of standards provided with the kit. The cAMP level in each well was normalized to total protein present in each well. Protein levels were measured using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, Illinois, USA). For each experiment, the standards were assayed in parallel Statistics Experiments were repeated at least three independent times with the specific number of repeats indicated in the individual figure legends. One-way Analysis of variance (ANOVA) and post- hoc Student-Newman-Keuls (SNK) tests and two-tailed Student’s t-test were performed with the SigmaPlotH 11 software (SPSS Inc.). Data were considered statistically significant when the probability value (P) was less than 0.05. Results D1 Dopamine Receptor is Post-transcriptionally Regulated during Postnatal Mouse Brain Development Previous studies in rats and humans have suggested that the D1 dopamine receptor exhibits post-transcriptional regulation during brain development [6,11,12]. Figure 1 shows the postnatal expression profile of D1 receptor mRNA and protein during postnatal brain development in mice. There is no significant difference in the expression of D1 receptor mRNA between day of birth (P0) through postnatal day 14 (P14) (Fig. 1A and 1C); in contrast, the relative expression of D1 receptor protein is low at P0 and begins to increase at P7 with a significant increase in expression on P14 (Fig. 1B and 1D). The specificity and sensitivity of the TaqManH probe and monoclonal antibody used to detect D1 receptor expression is shown in Figure S1A–C. The D1 dopamine receptor is glycosylated and the multiple bands in the gel represents various glycosylated forms of the D1 receptor. Together the results in Figure 1 suggest that D1 receptor expression is post-transcriptionally regulated during mouse brain development with a significant upregulation of mature D1 receptor protein on P14. D1 Dopamine Receptor is Post-transcriptionally Regulated in the Mouse CAD Catecholaminergic Cell Line The CAD cell line, derived from the CATH.a catecholamin- ergic cell line, is a mouse neuronal cell line that undergoes reversible morphological differentiation upon serum withdrawal (Fig. 2A and 2B) [14]. We have previously shown that the CAD cells express endogenous D1 receptor mRNA that yields functional D1 receptor protein [8]; however, compared to striatal tissue, the expression of D1 receptor protein is relatively low (Figure S2D). This suggests that D1 dopamine receptor is post-transcriptionally regulated in nondifferentiated CAD cells. Furthermore, consistent with our previous studies [8], the endogenous D1 dopamine receptor mRNA in CAD cells is significantly upregulated upon differentiation induced by serum withdrawal; however, the increase in D1 receptor mRNA is not accompanied by a concomitant increase in D1 receptor protein (Fig. 2C). The results suggest that the endogenous D1 receptor expression is post- transcriptionally regulated in non-differentiated and differentiated CAD cells, providing a convenient expression system to determine the molecular mechanisms underlying D1 receptor post-transcrip- tional regulation. The 39untranslated Region of D1 Receptor mRNA is Necessary and Sufficient for Post-transcriptional Regulation of D1 Receptor Expression To determine the post-transcriptional regulatory mechanisms of D1 receptor expression, we cloned a 6.4 kb upstream D1 receptor promoter region that included the 59 untranslated region (UTR) of PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49288