We name this pattern of labeling DAT-Type III (Fig. 5 C ).
We name this pattern of labeling DAT-Type II (Fig. 5 B ). Overall, the immunoprecipitate was paler in axons with DAT-Type II than in those with DAT-Type I immunolabeling.
In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool.
A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary.
We name this pattern of labeling DAT-Type I (Fig. 5 A ).
A similar but non-significant trend was seen for terminals labeled with anti-DAT antibody (Fig. 1F).
We made several attempts to label EL2-CCPGCC DAT using Lumio brand (Invitrogen) FlAsH reagent, using both standard approaches as well as modified protocols that use extended washes with BAL reagent to reduce non-specific FlAsH binding.
GFP-tagged DAT may not behave identically to the wildtype DAT, and has the disadvantage of labeling the entire cellular DAT population, limiting the ability to examine DAT surface dynamics specifically.
For comparison, neurons that were labeled by both TH and DAT antibodies, and non-immunoreactive tissue within the VTA, were also collected and analyzed by PCR.
While our data demonstrate that an extracellular tetracysteine-tag is well tolerated by DAT, they do not demonstrate whether this DAT construct can be labeled with biarsenic dyes.
Thin horizontal sections (35 μm) were cut in a cryostat at −21 to −22 °C and labeled together with antibody for tyrosine hydroxylase (TH; 1 1000, Millipore, Temecula, CA) and the DA transporter (DAT; 1 400, Synaptic Systems, Göttingen, Germany).
