Fig 1: Regulation of dopamine (DA)-mediated activation of the Golgi-localized D1DR in striatal neurons by OCT2.(a) Representative medium spiny neurons (MSN) expressing Snap-D1DR and Nb6B9-GFP at indicated times after 10 µM DA addition. DA stimulates D1DR activation at the Golgi in MSNs (n = 22 cells, Pearson’s coefficient = 0.67, six biological replicates). Arrow indicates active D1DR at plasma membrane; Arrowhead indicates active D1DR at Golgi membrane; Scale bar = 10 µm. (b) Representative MSN cell expressing Snap-D1DR and Nb6B9-GFP, pretreated with 100 µM imipramine for 15 min, before and after 10 µM DA addition. Inhibition of OCT2 blocks Golgi-localized D1DR activation at MSN n = 18 cells, Pearson’s coefficient = 0.38, six biological replicates but SKF81297 can still reach the Golgi membranes and activate D1DR Golgi pool (n = 6 cells, Pearson’s coefficient = 0.75, four biological replicates). Arrow indicates active D1DR at plasma membrane; Arrowhead indicates active D1DR at Golgi membrane; scale bar = 10 µm. (c) Quantification of Nb6B9-GFP recruitment at Golgi upon 10 µM DA stimulation in MSNs cells pretreated with OCT2 inhibitor; normalized fluorescence intensity of Nb6B9-GFP relative to Snap D1DR at Golgi (n = 12 and 7, respectively, five biological replicates). (d) Representative MSN expressing Snap-D1DR and Nb37-GFP before and after 10 µM DA addition. DA stimulates G protein activation at the Golgi in D1DR-expressing MSNs (n = 10 cells, Pearson’s coefficient = 0.34 and 0.62 before and after 10 µM DA stimulation, six biological replicates). Arrowhead indicates active Gs at Golgi membrane; right panels show zoomed images of insets for Snap-D1DR, Nb37-GFP, and the Golgi marker (GM130). Scale bar = 10 µm.
Fig 2: OCT2 facilitated dopamine (DA) transport to the Golgi-localized D1DR.(a) Representative HeLa cell expressing Snap-D1DR, Nb6B9-GFP, and GalT-mRFP expression pretreated with 100 µM imipramine for 15 min, before and after 10 µM DA addition. Inhibition of OCT2 blocks Golgi-localized D1DR activation but SKF81297 can still reach the Golgi membranes and activate D1DR Golgi pool (n = 30 cells, Pearson’s coefficient = 0.2 and 0.68 after DA and SKF addition, respectively, three biological replicates). Arrow indicates active D1DR at plasma membrane; arrowhead indicates active D1DR at Golgi membrane; Scale bar = 10 µm. (b) Quantification of Nb6B9-GFP recruitment at Golgi upon 10 µM DA stimulation in HeLa cells pretreated with 10 and 100 µM imipramine, 10 µM corticosterone, and (c) after 10 µM SKF81297 addition; normalized fluorescence intensity of Nb6B9-GFP relative to Snap D1DR at Golgi (n = 30, three biological replicates). (d) Representative HeLa cell expressing Snap-D1DR, miniGs-mApple, and SLC22A2 shRNA-B-GFP, before and after 10 µM DA addition. SLC22A2 shRNA blocks Golgi-localized D1DR activation. 10 µM SKF81297 addition activates D1DR at the Golgi (n = 16 cells, Pearson’s coefficient = –0.05 and 0.74 after DA and SKF addition, respectively, three biological replicates). (e) Detection of OCT2 expression in HeLa cells expressing different shRNAs by Western blot. (f) Quantification of D1DR activation at the Golgi in HeLa cells expressing scramble or SLC22A2 shRNAs upon addition of 10 µM DA; normalized fluorescence intensity of Nb6B9 at Golgi relative to Snap-tagged-D1DR at Golgi. (g) Quantification of D1DR activation at the Golgi in HeLa cells expressing SLC22A2 shRNA-B and D upon addition of 10 µM SKF81297.
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