Figure 3. Neurotransmitter transporters associate with β2‐adaptin and the SAC proteins MAD2, p31^comet, and BubR1, which are expressed in raphe nuclei. Effect of MAD2 depletion on SERT interactions.

• A, B
  Immunofluorescence of p31^comet and BubR1 in mouse dorsal raphe. White boxes indicate the magnified sections in the lower panels. Scale bars represent 100 and 25 μm in the upper and lower panels, respectively.
• C
  Co‐immunoprecipitation experiments using lysates from HEK‐293 cells stably expressing the indicated YFP‐tagged transporters. Eluates (20%) as well as 0.1% total cell lysate were analyzed by immunoblotting. The immunoblot for mouse anti‐β2‐adaptin was subsequently used for incubation with rabbit anti‐GFP primary and IRDye 800CW anti‐rabbit secondary antibody. IRDye signals were detected using the LI‐COR Odyssey CLx imaging system. Densitometric signals were quantified using ImageJ software.
• D–H
  HEK‐293 cells stably expressing YFP‐hSERT were transfected with indicated siRNAs (“C”—ctrl siRNA; “M”—MAD2 siRNA) and subjected to co‐immunoprecipitation. All signals were normalized to YFP‐SERT. Results were compared using unpaired two‐tailed t‐tests; ***P < 0.001, ****P < 0.0001, ns—not significant; calculated P‐values are indicated; error bars represent SD of seven (BubR1), eight (β2‐adaptin), five (p31^comet), and eight (HSP70) independent biological replicates.

Source data are available online for this figure.