PICK1–AP2 and PICK1–dynamin interactions are transiently enhanced after NMDAR stimulation by calcineurin activity. (A) NMDAR stimulation causes an increase in PICK1–clathrin colocalization. Hippocampal neurons expressing dsRed-clathrin and PICK1 shRNA + GFP-WT-PICK1 were exposed to NMDA for 3 min and then returned to normal medium for 4 min. Representative STED images of dendrites are shown. Bar, 1 µm. Graph shows PICK1 colocalization with clathrin (Manders coefficient; n = 3 independent experiments [11–16 cells per condition in total]; *, P < 0.05; Student’s two-tailed t test). (B) NMDAR stimulation transiently increases PICK1 interaction with AP2 and dynamin. Neurons were exposed to NMDA and then returned to normal medium. Cell extracts were prepared for immunoprecipitation with anti-PICK1 antibodies at the indicated time points after the end of NMDA application. Proteins were detected by Western blotting. Graphs show quantification of PICK1–AP2 and PICK1–dynamin binding (n = 5 independent experiments; *, P < 0.05; one-way ANOVA followed by Tukey’s test). (C) Calcineurin activity is required for the NMDA-induced increase in PICK1–AP2 and PICK1–dynamin interactions. Neurons were treated as in B, except that cells were exposed to 10 µM cyclosporin A 1 h before NMDA application. Graphs show quantification of PICK1–AP2 and PICK1–dynamin binding (n = 8 independent experiments; *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Tukey’s test; values are means ± SEMs).