Endogenous VAMP2 localizes predominantly to EEA1 or Rab11 compartments. (A) Representative confocal slice of a FLAG-SpH-MOR expressing cell showing colocalization of FLAG (magenta) and SpH (green) after 20 min 12.5 µM DAMGO treatment. Scale bar = 10 µm, and a line plot across a denoted section (yellow square and enlarged) of the representative slice. (B–D) Representative confocal slices of PC12 cells immunostained for VAMP2 (magenta) and EEA1 (B), Rab11 (C), or Rab7 (D; all in green). Example spots showing colocalization are marked with yellow arrows. Scale bar = 10 µm. Corresponding line plots across the denoted sections (yellow squares and enlarged) of the representative slices are shown for each image. (E) Percentage colocalization calculated using an automated object-based method, shown as colocalized spots normalized to either total SpH spots (/SpH) or total FLAG spots (/FLAG) from multiple image stacks (±SD, n = 20 from two independent coverslips). (F–H) Percent colocalization calculated as above for EEA1 (F), Rab11 (G), or Rab7 (H) are shown as colocalized spots normalized to total VAMP2 spots (/VAMP2) from multiple image stacks (±SD, n = 10 from two independent coverslips for each marker). “+” in the Tukey plots from E–H represent mean.