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Fig. 3. Rheb C180S expression is not associated with mTORC1. (A) Primary rat hippocampal neurons were transfected with plasmids for EGFP fusion proteins of Rheb WT, Rheb C180S and control vectors at E18 and stained after 3 d.i.v. with GFP (green), pmTOR (phospho-mTORSer2448) (red), tau (blue) antibodies and Hoechst (white). The scale bar is 2.5 µm. (B) The percentages for the pmTOR intensity level in the cell body and the axon (mean±S.E; *and #, *p<0.05 vs. control, ***p<0.0001, #p<0.05 vs. Rheb WT, ###p<0.0001 with one-way ANOVA and Tukey post hoc test, compared with control; n>50). (C) DMSO or rapamycin (200 nM) were added to the neurons for 2h after transfected neuron plating and stained after 3 d.i.v. with GFP (green) antibodies. Arrowheads indicate terminal ends of the axon. The scale bar is 50 µm. (D) The length of GFP-positive axons (mean±S.E.; *and #, *p<0.05 vs. control, #p<0.05 vs. control+rapamycin with one-way ANOVA and Tukey post hoc test, compared with control; n>25). (E) Primary rat hippocampal neurons were transfected with plasmids for EGFP fusion proteins of Rheb D60I, Rheb C180S, Rheb D60I+C180S or control vector constructs at E18 and stained and photographed on 3 d.i.v. for GFP (green), tau (red, axonal marker) and MAP2 (blue, dendrite marker) along with a Hoechst staining (white, nucleus). Arrowheads indicate terminal ends of the axon. The scale bar is 50 µm. (F) The length of GFP-positive axons (mean±S.E.; *and #, *p<0.05 vs. control, ***p<0.0001, #p<0.05 vs. Rheb D60I, ###p<0.0001 with one-way ANOVA and Tukey post hoc test, compared with control; n>25).
Exp Neurobiol 2019;28:172~182 https://doi.org/10.5607/en.2019.28.2.172
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