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 - PowerPoint PPT Presentation
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Experimental Date: 10-04-13 • 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 To examine D2 receptor trafficking modulated by RGS2 expression or f
Transcript
Experimental Date: 10-04-13
• 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
Purpose: To examine D2 receptor trafficking modulated by RGS2 expression or function
Confocal Microscopic Images
• Objective: oil (?) 63x (?)• Ruler:
Control
Veh
Membrane Cytosol Overlay
Quin 5 min
Quin 15 min
DN RGS2
Veh
Membrane Cytosol Overlay
Quin 5 min
Quin 15 min
WT RGS2
Veh
Membrane Cytosol Overlay
Quin 5 min
Quin 15 min
Control
DN
WT
Veh Treatment
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
Experimental Date: 10-10-13
• 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
Purpose: To examine D2 receptor trafficking modulated by RGS2 expression or functionControl, WT, DN, siRNAs
Experimental Date: 10-14-13
• 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
Purpose: To examine colocalization of D2 receptor and RGS2 upon quinpirole tratment
