Figure 3.

CRISPR–Cas9–mediated Rab43KO suppresses the dendritic and postsynaptic transport of α_2B-AR, but not M3R, in hippocampal neurons.A, CRISPR–Cas9–mediated depletion of Rab43. HEK293 cells were transfected with GFP-tagged rat Rab43 together with CRISPR–Cas9 control vectors or KO plasmids targeting mouse Rab43. GFP–Rab43 expression was revealed by Western blotting using GFP antibodies. Similar results were obtained in three experiments. B and C, effects of Rab43KO on the dendritic expression of α_2B-AR (B) and M3R (C). Hippocampal neurons were cultured on 12-well dishes and transfected with 250 ng of α_2B-AR–CFP (B) or M3R–CFP (C) together with 500 ng of control or Rab43KO plasmids for 48 h. D, quantitative data shown in panel B and C. E and F, effects of Rab43KO on the postsynaptic expression of α_2B-AR (E) and M3R (F). G, quantitative data shown in panels E and F. H and I, hippocampal neurons were cultured on 12-well dishes and transfected with α_2B-AR–YFP (H) or M3R–YFP (I), RFP-PSD95, and control or Rab43KO plasmids for 48 h. J, receptor expression at postsynapses was measured by using PSD95 as a marker. Bars represent the mean ± SD (n = 15–20 neurons in panel D, n = 75–82 spines in panel G, and n = 62–70 spines in panel J in at least five independent experiments). ∗p < 0.05 relative to the respective control. In panels B and C, the scale bar represents 50 μm, and in panelsE, F, H, and I, the scale bar represents 5 μm. α₂-AR, α₂-adrenergic receptor; CFP, cyan fluorescent protein; M3R, M3 mAChR; YFP, yellow fluorescent protein.