Figure 3.

TNR recycles in neurons over ~3 days. (A) A schematic of an assay to assess TNR recycling. (1) Cultured hippocampal neurons were pulsed with recombinant His-tagged TNR, which was then allowed to potentially internalize and recycle for a period of 0–3 days (2). After the incubation period, the neurons were immediately fixed (3), or fixed following treatment with proteinase K to strip away all surface-bound recombinant TNR molecules (3’). Following permeabilization treatment, the neurons were immunostained using antibodies against the His-tag to visualize all recombinant TNR (4), or the internalized recombinant TNR only (4’). (B) Immediately after pulsing the neurons with recombinant TNR, the staining was visibly reduced by the surface stripping, indicating that the majority of the molecules were surface-bound. One day after the pulse, the signal was similar for non-stripped and stripped neurons, indicating that most molecules had been internalized. Three days after the pulse, surface stripping visibly reduced the staining once again, indicating that a portion of recombinant TNR molecules had recycled back to the surface. Blue arrowheads indicate labeledTNR in neurites. Scale bar = 10 μm. Statistical significance was evaluated with repeated-measures one-way ANOVA (F_{1.044, 2.088} = 28, 6, *p = 0.03), followed by Fisher’s LSD (“0 days” vs. “1 day”: **p = 0.002; “1 day” vs. “3 days”: *p = 0.027; “0 days” vs. “3 days”: p = 0.775). N = 3 independent experiments. In the plot, lines represent the means, shaded areas represent the SEM, and dots represent individual experiments. Adapted from Dankovich et al. (2021) with permission from Springer Nature (http://creativecommons.org/licenses/by/4.0/).