Experimental Date: 10-04-13

1. Experimental Date: 10-04-13 Purpose: To examine D2 receptor trafficking modulated by RGS2 expression or function • N2A-hD2S stable cell line with eGFP expression • Cells were transiently transfected with empty vector, RGS2N149A (DN) and WT RGS2 construct • 48 hrs after the transfection, cells were treated with Veh or quinpirole (1µM) for 5 min or 15 min • Immunocytochemistry was performed to examine the surface and cytosolic expression of D2 receptors • Block for 20 min on ice • Surface labeling: rabbit anti-D2R (1:50 dilution, Santa Cruz) for 90 min on ice • Secondary labeling: goat anti-rabbit 594 (1:250, Invitrogen) for 1 hr on ice • Fix for 15 min on ice • Permeabilize: for 10 min on ice • Intracellular labeling: mouse anti-D2R (1:100 dilution, Santa Cruz) for 1 hr at RT • Secondary labeling: goat anti-mouse 405 (1:300, Invitrogen) for 45 min at RT • Prolong gold without DAPI

2. Confocal Microscopic Images • Objective: oil (?) 63x (?) • Ruler:

3. Control Membrane Cytosol Overlay Veh Quin 5 min Quin 15 min

4. DN RGS2 Membrane Cytosol Overlay Veh Quin 5 min Quin 15 min

5. WT RGS2 Membrane Cytosol Overlay Veh Quin 5 min Quin 15 min

6. Veh Treatment Control DN WT

7. Data • A bar graph for the basal (veh treatment) D2R (red/blue) surface expression for control, WT and DN constrcuts (change the intensity of the red or blue) • Representative confocal images • A bar graph for the effect of quinporle dose on surface D2R expression (red/blue) for each construct

8. Experimental Date: 10-10-13 Purpose: To examine D2 receptor trafficking modulated by RGS2 expression or function Control, WT, DN, siRNAs • N2A-hD2S stable cell line with eGFP expression • Cells were transiently transfected with empty vector, RGS2N149A (DN) and WT RGS2 construct • 48 hrs after the transfection, cells were treated with Veh or quinpirole (1µM) for 5 min or 15 min • Immunocytochemistry was performed to examine the surface and cytosolic expression of D2 receptors • Block for 20 min on ice • Surface labeling: rabbit anti-D2R (1:50 dilution, Santa Cruz) for 90 min on ice • Secondary labeling: goat anti-rabbit 594 (1:250, Invitrogen) for 1 hr on ice • Fix for 15 min on ice • Permeabilize: for 10 min on ice • Intracellular labeling: mouse anti-D2R (1:100 dilution, Santa Cruz) for 1 hr at RT • Secondary labeling: goat anti-mouse 405 (1:300, Invitrogen) for 45 min at RT • Prolong gold without DAPI

9. Experimental Date: 10-14-13 Purpose: To examine colocalization of D2 receptor and RGS2 upon quinpirole tratment • N2A-hD2S stable cell line with eGFP expression • Cells were treated with Veh or quinpirole (1µM) for 5, 15 or 30 min • Immunocytochemistry was performed to examine the surface D2 receptor and surface or cytosolic expression of RGS2 protein • 1st set: surface D2R and RGS2 • Block for 20 min on ice • Surface labeling of rabbit anti-D2R (1:50, santa cruz), mouse anti-RGS2 (1:100, Sigma) for 90 min on ice • Secondary labeling: goat anti-rabbit 594, goat anti-mouse 405 for 1 hr on ice • Fix and add prolong gold without DAPI • 2nd set: Surface D2R and total RGS2 • Block for 20 min on ice • Surface labeling of D2R (rabbit anti-D2R, 1:50, 1 hr on ice), secondary: goat anti-rabbit 594 (1hr on ice) • Fix and permeabilize • Labeling of total RGS2: mouse anti-RGS2 (1:100, 1hr), secondary: goat anti-mouse 405 (1:250, 45 min) at room temperature • Prolong gold without DAPI • 3rd set: total D2R and RGS2 • Fix and permeablize • Primary antibodies: rabbit anti-D2R and mouse anti-RGS2 (1 hr at RT) • Secondary: goat anti-rabbit 594 and goat anti-mouse 405 (1 hr at RT) • Prolong gold without DAPI