Ab2914

N2A-D2R cells were treated with vehicle or quinpirole (1 μM, 5 or 30 min). Then surface D2Rs were labeled with a mouse anti-D2R antibody (sc-5303, Santa Cruz) at 4°C followed by goat anti-mouse Alexa Fluor 594. Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton-X. Cytoplasmic β-arrestin was subsequently labeled with rabbit anti-β-arrestin (ab2914, Abcam) followed by goat anti-rabbit Alexa Fluor 405 antibodies. Colocalization between β-arrestin and surface D2Rs was quantified using Coloc2 software (FIJI) as previously described (Cleghorn et al., 2015 (link)) and Pearson R correlation coefficients were generated for individual cells. Pearson R values were averaged across cells for each group and data were presented relative to the level of colocalization between β-arrestin and surface D2Rs in control cells with vehicle treatment.

Briefly, N2A-D2R cells were stimulated with vehicle or quinpirole (1μM, 5 or 30 min) followed by fixation, permeabilization and blocking with 6% horse serum in PBS. Cytoplasmic β-arrestin and Rab5 (a marker for the early endosome), transferrin receptors (a marker for the recycling endosome) or LAMP1 (a marker for the late endosome) were probed with the following primary antibodies: rabbit anti-β-arrestin (ab2914, Abcam), mouse anti-Rab5 (sc-46692, Santa Cruz), goat anti-Transferrin (ab166929, Abcam), and mouse anti-LAMP1 (sc-17768, Santa Cruz). Then cells were incubated with rabbit anti-mouse Alexa 405, mouse anti-rabbit Alexa 555 and donkey anti-goat Alexa 633, respectively. Colocalization of β-arrestin with Rab5, transferrin receptors or LAMP1 was calculated using Coloc2 software (FIJI). Data are presented relative to the level of colocalization between β-arrestin and each endocytic marker in control cells with vehicle treatment.