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Fig. 4 | Molecular Brain

Fig. 4

From: A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity

Fig. 4

Synergistic activation of mTORC1 by Rheb and LE-associated mTOR. A Association of Rheb with LEs. DIV 13 hippocampal neurons were transfected with mCherry-Rheb and GFP-Rab7 and fixed three days later. Shown is an epifluorescence image of a dendritic region. A majority of Rheb positive structures are Rab7 positive (examples are depicted with arrowheads). Bar, 10 µm. B Overexpression of Rheb in neurons activates mTORC1 pathway. DIV 13 neurons were transfected with WT or Q64L mCherry-Rheb and 4 days’ later immunofluorescence for P-pS6 was performed with or without 3 h pre-incubation with 100 nM rapamycin. Shown is P-pS6 intensity of transfected neurons expressed as percentage of the mean fluorescence measured in control neurons (transfected with empty mCherry vector) from two independent experiments with 40–50 neurons per conditions. C Epifluorescence images of hippocampal neurons transfected with Flag-Raptor-Rheb15-TS and labeled with anti-LAMP1 and anti-Flag antibodies. Regions boxed in C are shown at higher magnification on the right panel. Most of LAMP1-positive structures are also positive for Flag-Raptor-Rheb15-TS. Bar, 10 µm. D DIV14 neurons were transfected with mCherry or mCherry-Rheb, Flag-Raptor-Rheb15-TS or myc-Raptor as indicated and 4 days later labeled with anti-P-pS6 and anti-Flag or anti-myc antibodies. Neurons expressing the constructs were imaged for P-pS6 intensity. Flag-Raptor-Rheb15-TS alone had no effect on P-pS6 levels but increased mTORC1 activity when combined with Rheb overexpression (N = 30 images from two independent experiments)

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