D1 Receptor Expression Is Regulated by MicroRNA Figure 2. Endogenous D1 dopamine receptor exhibits post-transcriptional regulation in the mouse CAD catecholaminergic cell line. (A) Representative images of non-differentiated (ND) and (B) differentiated (D) CAD cells. (C) Cumulative data showing relative D1 receptor mRNA and protein in non-differentiated (ND) and differentiated (D) CAD cells. The mRNA levels were measured using gene-specific TaqManH probes and the D1 receptor protein levels detected using the rat monoclonal antibody. The D1 receptor mRNA levels were normalized to GAPDH mRNA levels and D1 receptor protein was normalized to total protein. The inset shows a representative gel with D1 receptor mRNA and D1 receptor protein in non- differentiated (N) and differentiated (D) CAD cells. The plotted bars represent the mean values 6 s.e.m. (n = 5). *P,0.001, significant difference in D1 receptor mRNA levels between non-differentiated and differentiated CAD cells, two-tailed pair-wise Student’s t-test. doi:10.1371/journal.pone.0049288.g002 Figure 3. Reporter gene regulated by the mouse D1 receptor promoter and 39UTR exhibits post-transcriptional regulation. (A) Schematic representation of the two EGFP-reporter constructs used. One construct (6.4 D1) has the 6.4 kb D1 receptor promoter and the heterologous SV40 39UTR; the other construct (6.4 D1+39UTR) has the 6.4 kb D1 receptor promoter and the 1277 bp D1 39UTR. (B) Representative real-time RT-PCR gel using the SYBERH green format showing the mRNA levels of EGFP reporter, BAP-FlagTM (transfection control), endogenous D1 receptor and 18S ribosomal RNA (internal control) in non-differentiated (ND) and differentiated (D) CAD cells harvested 48 hours after transient transfection of the 6.4D1 or the 6.4 D1+39UTR reporter constructs. The PCR reactions were stopped in the exponential phase and the products run out on the gel. (C) Cumulative data showing normalized EGFP reporter mRNA levels from ND and D CAD cells transfected with the 6.4 D1 or the 6.4 D1+39UTR constructs. The EGFP mRNA levels were normalized to co-transfected BAP-FlagTM mRNA and the endogenous 18S ribosomal RNA. The bars represent the mean values 6 s.e.m. (n = 7). *P,0.004, significant difference between ND and D CAD cells transfected with either reporter constructs, One Way ANOVA, post hoc SNK test. (D) Representative western blot gel showing the protein levels of EGFP reporter, BAP-FlagTM (transfection control) in ND and D CAD cells harvested 48 hours after transient transfection of the 6.4 D1 or 6.4 D1+39UTR reporter constructs. (E) Cumulative data showing normalized EGFP reporter protein levels from ND and D CAD cells transfected with the 6.4 D1 or the 6.4 D1+39UTR constructs. The EGFP protein levels were normalized to co-transfected BAP-FlagTM protein and total protein levels in the lysate. The bars represent the mean values 6 s.e.m. (n = 7). *P,0.001, significant difference between CAD cells transfected with the 6.4 D1 or the 6.4 D1+39UTR constructs in either ND or D CAD cells, #, P,0.001, significant difference between ND and D CAD cells transfected with the 6.4 D1 reporter construct, One Way ANOVA, post hoc SNK test. doi:10.1371/journal.pone.0049288.g003 PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49288