Fig. 2. Rheb C180S expression induces phosphorylation of 4EBP1. (A) Neuro2A (N2A) cells were transfected with EGFP co-expressing Rheb WT, Rheb C180S or control vectors for 48h before assaying for the expression of pS6K (phospho-S6K, Ser240/244), p4EBP1 (phospho-4EBP1 Ser65, Thr37/36) and total S6K, 4EBP1 via western blot with the percentages of band intensities shown. (B) Primary rat hippocampal neurons were transfected with plasmids for EGFP fusion proteins of Rheb WT, Rheb C180S or control vectors at E18 and stained on 3 d.i.v. with GFP (green), p4EBP1 (Thr37/46) (red), tau (blue) antibodies and Hoechst (white). The scale bar is 2.5 µm. (C) Percentages for the p4EBP1 intensity levels in the cell body and in the axon (mean±S.E.; *and #, *p<0.05 vs. control, ***p<0.0001, #p<0.05 vs. Rheb WT with one-way ANOVA and Tukey post hoc test, compared with control; n>35). (D) Primary rat hippocampal neurons were co-transfected with plasmids mCherry co-expressing Rheb C180S, Rheb WT or control vectors at E18 and stained on 3 d.i.v. with GFP (green), mCherry (red) antibodies (upper panel). Primary rat hippocampal neurons were co-transfected with plasmids for EGFP co-expressing 4EBP1 F113A with mCherry co-expressing Rheb C180S, Rheb WT or control vectors at E18 and stained on 3 d.i.v. with GFP (green), mCherry (red) antibodies (lower panel). Arrowheads indicate terminal ends of the axon. The scale bar is 50 µm. (E) The percentages for the axonal length for each group of transfectants (mean±S.E.; *and #, *p<0.05 vs. control, ***p<0.0001, #p<0.05 vs. 4EBP1 F113A with one-way ANOVA and Tukey post hoc test, compared with control; n>35).
© Exp Neurobiol
