Figure 2. TrkB activation, co-localization with GABA_ARα1, and GABA_ARα1 internalization.

Panel A: surface (left) or total (right) GABA_ARα1 expression (green), TrkB expression (red), or the merge between the two (yellow). Panel B: an immunoblot, with phospho-TrkB (top, arrow is p-TrkB) and total TrkB (bottom), with vehicle vs. BDNF treatment at 5 min, 20min, and 2hrs after BDNF addition. Panel C: Confocal microscopy analysis of surface (green) and internalized (red) GABA_ARα1 subunits. Untreated neurons have abundant surface, but undetectable internal GABA_ARα1. BDNF (5, 10, or 20 minutes) treated cultures show internalized alpha 1 (red) in neuronal soma (5 min) and dendrites (10 and 20 min) after BDNF treatment. Arrows indicate internalized GABA_ARα1 subunits. Panel D: Biotinylation studies showing that the apparent internalization of GABA_ARα1 seen with antibody feeding techniques is confirmed with biochemical analyses of surface receptor localization. Western blot analyses is shown of biotin-precipitated, surface GABA_ARα1, total GABA_ARα1 from cell homogenate, and total GADPH as a loading control. Panel E: Quantification of surface:total GABA_ARα1 ratio following vehicle (control) or 10–20 minutes of BDNF treatment using the biotinylation surface-labeling technique. (Mean ± SEM, * p < 0.01, relative to control).